Switchable surfaces for biomedical...
Transcript of Switchable surfaces for biomedical...
Switchable Surfaces for Biomedical
Applications
by
Eleonora Cantini
A thesis submitted to
The University of Birmingham
for the degree of
DOCTOR OF PHILOSOPHY
School of Chemical Engineering
College of Physical Sciences and Engineering
The University of Birmingham
May 2017
University of Birmingham Research Archive
e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
Abstract
Switchable oligopeptides, able to expose or conceal biomolecules on a surface, upon
the application of an electrical potential, represent a versatile tool for the development of
novel devices, presenting potential biomedical applications.
Recently, several studies have demonstrated the applicability of smart devices for the
control of protein binding and cellular response. In this work, a detailed analysis of the steric
requirements necessary to develop a mixed oligopeptide Self-Assembled Monolayer (SAM)
presenting an optimum switching ability will be described. The influence of both the SAM
components surface ratio and the switching unit length on the mixed SAMs switching
performance will be investigated.
The findings of this investigation will be used to develop for the first time a device, based
on electrically switchable oligopeptides, able to control the interaction between an antigen and
its relative antibody. The influence of the biological medium on the oligopeptide switching
ability will also be investigated.
Finally, an orthogonal functionalisation strategy will be investigated in detail, together
with a new platform able to promote human sperm cells adhesion. The possible obstacles
present in the process will be described and examined thoroughly.
The results of this research thesis will also represent the first building blocks towards
the development of glass-gold micropatterned surfaces able to control the calcium signalling
in human sperm cells. Hence, the work presented in this dissertation will have important
applications in the development of both new devices to control antibodies response and new
efficient methods to select human sperm cells for in-vitro fertilisation (IVF) treatments.
Dedicated to the memory of dear friend and colleague
Simone Basile (1988-2016)
Acknowledgments
My biggest thank you goes to my supervisor Professor Paula M. Mendes, for her fundamental
support and advice throughout my PhD, but also for being patient and helpful during my
writing-up. I would also thank my co-supervisor Professor Jon A. Preece, for his supervision,
help and support. This research work would have not been completed without the guidance
from you both.
Special thanks to Dr Jackson Kirkman-Brown, Dr Lorraine Frew and Dr Joao Correia for their
precious help during the second part of this research work.
I would also like to say “thank you” to the members of both the Preece and Mendes group past
and present: Dr James Bowen, especially for this help and support with the ellipsometer, Dr
Paul Yeung, for his help and support with surface chemistry and SPR instrumentation, Dr Alice
Pranzetti, Dr Parvez Iqbal Dr Alex Stevenson- Brown, Dr Minhaj Lashkor, Dr Nga Yip, Dr Aaron
Acton, Dr Lewis Hart, Dr Stefano Tommasone, Simone Basile, Monika Köpf, Philippa Mitchell,
Bárbara Santos-Gomes, Fatima Zia, Francia Allabush, Zarrar Hussain and both Greg O’Callaghan
and Dennis Zhao for their support during my PhD.
Special thanks go to my friend Dr Steve Della Mora, for his friendship, his patience and his
support during the whole duration of my thesis and for deterring me from giving up.
A big thank you also to Dr Peter Jervis, Dr Lucia Cardo, Dr Giacomo Volpe, Dr Claudio Zito, Javier
Jimenez, Chiara Busà, Simone Damiani and all the people I met during my three years at
University of Birmingham. You helped me to remain mentally healthy.
I cannot forget to say thank you to Dr Avril Day-Jones, Katharine Partington and all the people
I met since September, while I was trying to write a PhD thesis and training as a Science teacher
at the same time. Your support has been fundamental.
Finally, I would like to thank my parents for their support from Italy during my studies and to
my boyfriend Darren, for supporting, encouraging me, reminding me “never give up” and
especially teaching me what being patient mean. Thank you to Darren’s family too, for your
encouragements.
Without the people name above, this work would have not been completed. I will always be
grateful to you.
Table of Contents
Chapter 1 – An introduction to Bionanotechnology and Self-
Assembled Monolayers ................................................................... 1
1.1 Bionanotechnology .................................................................................................... 1
1.1.1 Surface Functionalisation for biosensors, cell sensing and diagnostic ........................ 1
1.1.2 Other materials used in biosensing ............................................................................. 3
1.2 Functionalisation approaches .................................................................................... 4
1.2.1 Top-down approach ..................................................................................................... 5
1.2.2 Bottom-up approach.................................................................................................... 7
1.3 Self-Assembled Monolayers ....................................................................................... 8
1.3.1 Surfactant .................................................................................................................... 8
1.3.2 Substrates and SAMs ................................................................................................. 10
1.3.2.1 Silane SAMs ...................................................................................................................... 10
1.3.2.2 Thiol SAMs ....................................................................................................................... 12
1.3.3 Mixed SAMs ............................................................................................................... 14
1.3.3.1 Unspecific protein adsorption .......................................................................................... 16
1.3.3.2 Specific protein adsorption .............................................................................................. 17
1.3.4 Dynamic SAMs ........................................................................................................... 20
1.3.5 Electrically-switchable SAMs ..................................................................................... 23
1.4 Application of switchable surfaces to the selection of sperm cells for in-vitro
fertilisation (IVF) techniques .......................................................................................... 30
1.5 Concluding remarks ................................................................................................. 32
1.6 PhD aim ................................................................................................................... 34
Chapter 2: Surface Characterisation Techniques ............................ 37
2.1 Contact Angle ...................................................................................................... 37
2.2 X-ray Photoelectron Spectroscopy ....................................................................... 40
2.3 Ellipsometry ........................................................................................................ 43
2.4 Fluorescence Microscopy .................................................................................... 45
2.5 Surface Plasmon Resonance (SPR) ....................................................................... 48
2.6 Electrochemical techniques ................................................................................. 53
2.6.1. Chronoamperometry ............................................................................................ 53
2.6.2. Linear Sweep Voltammetry .................................................................................. 54
2.6.3. Cyclic Voltammetry .............................................................................................. 57
Chapter 3 - Experimental Procedures and Protocols ....................... 61
3.1 Materials and Methods ............................................................................................ 61
3.1.1 Gold substrates .......................................................................................................... 61
3.1.2 Glass substrates ......................................................................................................... 61
3.1.2 Silicon substrates ....................................................................................................... 61
3.2 Chemicals ................................................................................................................ 62
3.3 Experimental Procedures ......................................................................................... 63
3.3.1 Surface Preparation ................................................................................................... 63
3.3.1.1 Cleaning of gold and glass surfaces ................................................................................. 63
3.3.1.2 Preparation of Biotin-2KC:TEGT, Biotin-4KC:TEGT and Biotin-6KC:TEGT mixed Self-
Assembled Monolayers (SAMs) on gold substrates ..................................................................... 64
3.3.1.3 Preparation of silane-PDL on glass substrates and silicon wafer .................................... 64
3.3.2 Surface characterisation ............................................................................................ 65
3.3.2.1 Contact angle ................................................................................................................... 65
3.3.2.2 Ellipsometry ..................................................................................................................... 66
3.3.2.3 Surface Plasmon Resonance ............................................................................................ 66
3.3.2.4 X-ray Photoelectron Spectroscopy ................................................................................... 68
3.3.2.5 Force Field Test ................................................................................................................ 69
3.2.2.6 Computational details ..................................................................................................... 71
3.3 Preparation and Analysis of Sperm Cells ................................................................... 71
3.3.1 Sperm Cells Incubation and labelling ......................................................................... 71
3.3.2 Sperm Cells Counting ................................................................................................. 72
3.3.3 Perfusion Chamber .................................................................................................... 72
3.3.4 Fluorescence Microscopy ........................................................................................... 73
Chapter 4 - Study of the effect of switching unit length on switching
ability............................................................................................ 74
4.1 Introduction ............................................................................................................ 75
4.2 Objectives ............................................................................................................... 78
4.2 Results and discussion ............................................................................................. 79
4.2.1 Formation of mixed biotin-4KC:TEGT SAMs .............................................................. 79
4.2.2 XPS characterisation of mixed biotin-4KC:TEGT SAMs .............................................. 80
4.2.3 Investigation on biotin-4KC:TEGT binding capacity and switching efficiency ........... 88
4.3 Study on the influence of switching unit length on switching efficiency .................... 93
4.3.1 XPS characterisation of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs ............ 93
4.3.2 SPR analysis of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs switching
properties ............................................................................................................................ 98
4.2.5 Molecular Dynamics Simulations............................................................................. 101
4.4 Conclusions ........................................................................................................... 108
Chapter 5 - Study of the switching properties of progesterone-C7-
4KC:EG6OH mixed SAMs ............................................................. 110
5.1 Introduction .......................................................................................................... 110
5.2 Objectives ............................................................................................................. 114
5.3 Results and Discussion ........................................................................................... 115
5.3.1 Formation of mixed progesterone-C7-4KC:EG6OH mixed SAMs ............................. 115
5.3.2 XPS characterisation of progesterone-C7-4KC:EG6OH mixed SAMs ....................... 116
5.3.2.1 Progesterone-C7-4KC:EG6OH 1:10 solution ratio .......................................................... 116
5.3.2.2 Progesterone-C7-4KC:EG6OH 1:40 solution ratio .......................................................... 119
5.3.2.3 Progesterone-C7-4KC:EG6OH 1:100 solution ratio ........................................................ 122
5.3.3 Contact angle and ellipsometry characterisation of progesterone-C7-4KC:EG6OH
mixed SAMs ...................................................................................................................... 124
5.4 SPR analysis of the progesterone-C7-4KC:EG6OH mixed SAMs and anti-mouse
progesterone antibody ................................................................................................ 126
5.4.1 Testing the anti-mouse progesterone antibody specificity by SPR .......................... 126
5.4.2 SPR analysis of the switching capabilities of progesterone-C7-4KC:EG6OH mixed
SAMs in Phosphate Saline Buffer ...................................................................................... 128
5.4.3 SPR analysis of the switching capabilities of 1:40 progesterone-C7-4KC:EG6OH
mixed SAMs solution ratio in modified Earle’s Buffer Saline Solution containing 0.3% of
Bovine Serum Albumin ...................................................................................................... 133
5.4.4 SPR analysis of the switching capabilities of 1:40 progesterone-C7-4KC:EG6OH
mixed SAMs solution ratio in modified Earle’s Buffer Saline Solution containing 0.1% and
0% of Bovine Serum Albumin ............................................................................................ 136
4.5 Conclusions and Future Work................................................................................. 142
Chapter 6 – Orthogonal Functionalisation of Surfaces for Controlling
Sperm Cell Adhesion and Hyperactivation ................................... 144
6.1 Introduction .......................................................................................................... 144
6.2 Objectives ............................................................................................................. 147
6.3 Results and Discussion ........................................................................................... 147
6.3.1 Study of sperm cells attachment on poly-D-lysine and silane/poly-D-lysine layers and
surface characterisation ................................................................................................... 147
6.3.1.1 XPS characterisation of poly-D-lysine and silane/poly-D-lysine layers .......................... 149
6.3.1.2 Contact angle and ellipsometry analysis of poly-D-lysine and silane/poly-D-lysine layers
................................................................................................................................................... 153
6.3.1.4 Effect of different solvents on poly-D-lysine and silane/poly-D-lysine layers and sperm
cells attachment ........................................................................................................................ 155
6.3.2 XPS characterisation of clean glass and gold substrates ........................................ 159
6.3.3 XPS characterisation of each step performed on either plain gold or glass surfaces
.......................................................................................................................................... 163
6.3.3.1 First step: protective thiol on gold and glass ................................................................. 163
6.3.3.2 Second step: silane-PDL on gold and glass .................................................................... 168
6.3.3.3 Third step: protective thiol removal ............................................................................... 173
6.3.3.4 Fourth step: Progesterone-C7-4KC mixed SAM ............................................................. 179
6.3.4 Study of the effect of surface preparation steps on cell adhesion .......................... 185
6.4 Conclusions and Future Work................................................................................. 186
Chapter 7 – Conclusions and Future Work.................................... 189
7.1 Conclusions ........................................................................................................... 189
7.2 Future Work .......................................................................................................... 192
References .................................................................................. 195
List of Illustrations and Tables
Chapter 1
Figure 1.1 – Cartoon representation of top-down and bottom-up approach
Figure 1.2 – Schematic representation of the photolithographic process. The substrate covered
with a masking film (A) is coated with a photoresist (B). A mask is then collocated on the
photoresist film (C) to create the desired pattern after UV exposure (D) and etching (E). Finally,
the photoresist is completely removed by stripping, to obtain the desired patterned substrate
(F).
Figure 1.3 – Schematic representation of a surfactant molecule
Figure 1.4 – Cartoon representation of a silane SAM.
Figure 1.5 – Schematic of the three-steps process for SAMs formation. 1) Physisorption, 2)
chemisorption and 3) completion of SAM spatial orientation and packing.
Figure 1.6 – Schematic representation of the arrangement of dodecanethiol molecules on Au
(111) substrates to achieve maximum surface coverage. (a) Structural model of the hexagonal
arrangement of thiols (black hexagon) on gold and the area covered by each thiol molecule
(dashed-lined circles). (b) Cross−section of the SAM formed showing the alkane chains 30˚
tilting in the direction of their next−nearest neighbours.
Figure 1.7 – Cartoon representation of a mixed SAM
Figure 1.8 – Cartoon representation of biotin binding to two of the four binding sites of the
avidin family protein
Figure 1.9 – Schematic representation of an antigen-antibody system. Several antigens are
available for binding, but only one is specific for the antigen binding site on the antibody chains
Figure 1.10 – Schematic of the possible switches that can be obtained upon the application of
a stimulus. a) Chemical switch: the chemical composition of the monolayer varies when an
external stimulus is applied (e.g. a change in pH). b) Conformational switch: the chemical
composition of the monolayer remains the same, while its conformation varies when an
external stimulus is applied (e.g. application of an electrical potential).
Figure 1.11 – Illustration of the formation and switching of a LD-SAM by employing a precursor
molecule presenting a bulky head-group. Upon the application of an electrical potential the
monolayer can reverse its wettability by switching between hydrophilic and hydrophobic
states.
Figure 1.12 – Schematic representation of the DNA lever; a) lying on the surface when a positive
potential is applied and repelled when a negative potential is applied, fully exposing the
fluorescent dye. b) When a protein is linked to the DNA’s top end, the upward motion is slowed
and lags behind the bare lever. The dark yellow circle represents the Cy3 dye when the
fluorescence is quenched by the gold surface. The bright yellow circle corresponds to a high
fluorescence emission, due to the DNA molecule carrying the fluorophore being fully extended
on the surface.
Figure 1.13 – Schematic representation of the switching of Biotin-4KC:TEGT mixed SAM upon
the application of an electrical potential.
Figure 1.14 – Cartoon representation of the control of bacteria cells adhesion by switching a
MUA:MET mixed SAM.
Figure 1.15 - Molecular structures and related cartoons of the oligopeptide Biotin-4KC and the
triethylene glycol-terminated thiol (TEGT) used in the mixed SAMs.
Figure 1.16 – Cartoon representation of the switchable system (top), molecular structures and
related cartoons of the oligopeptide (progesterone-C7-4KC) and the hexaethylene glycol-
terminated thiol (EG6OH) used in the mixed SAMs (bottom).
Chapter 2
Figure 2.1 – Schematic representation of a contact angle goniometer
Figure 2.2 – Representation of contact angles (θ) formed by sessile liquid drops on a solid
surface. Representation of a) a hydrophilic surface and b) a hydrophobic surface.
Figure 2.3 – Schematic representation of a) advancing (θa) and b) receding contact angle (θr)
used in the dynamic contact angle measurements.
Figure 2.4 - Cartoon representation of a XPS apparatus
Figure 2.5 – Schematic diagram of XPS process for a photoelectron emitted from the core
energy level. The subsequent relaxation process of an electron from a higher energy level
(dashed arrow) fills the created vacancy (white circle), resulting in the emission of an Auger KLL
electron (dark green circle).
Figure 2.6 – Schematic representation of an ellipsometer.
Figure 2.7 – Schematic illustration of a fluorescence microscope setup.
Figure 2.8 - Kretschmann geometry of the Attenuated Total Reflection (ATR) method. θSP is the
angle at which the incident light is able to excite the surface plasmon wave (SPW) at the metal-
dielectric interface, d is the thickness of the metal surface (usually 50 nm) and εm and εd are
the dielectric constants of the metal and the dielectric, respectively.
Figure 2.9 – Illustration of the SPR sensing principle, using the Kretschmann optical
configuration. a) The SPR detects the angle of reflection of the light when no analyte is bound
to the SAM on the surface; b) when the analyte is present in solution, this comes into contact
with the target molecules on the surface. The binding causes a variation in the mass present
on the surface, therefore a shift in the angle of reflection (c) occurs, and a change in the
intensity of the reflected is recorded by the photodiode array detector. d) A sensorgram is
obtained by plotting the resonance angle signal against time, and it is used to monitor the
changes occurring on the surface.
Figure 2.10 – Illustration of a) chronoamperometry potential stepping and b) current variation
with time
Figure 2.11 – Series of linear sweep voltammograms recorded at different scan rate
Figure 2.12 – Changes in current response for voltammograms recorded at different scan rates.
Figure 2.13 – Schematic representation of the forward and back scans in cyclic voltammetry.
Figure 2.14 – Schematic representation of a cyclic voltammogram for a reversible single
electrode transfer reaction, in the case of a solution containing only a single electrochemical
reactant. Epc and ipc are the peak potential and peak current relative to the cathode,
respectively, whereas Epa and ipa are the peak potential and peak current relative to the anode,
respectively
Chapter 3
Figure 3.1 – Multistep route for the synthesis of triethylene glycol thiol (TEGT)
Figure 3.2 – Multistep route for the functionalisation of glass or silicon substrates with silane-
PDL layers
Figure 3.3 – Cartoon representation of the SPR electrochemical cell
Figure 3.4 - The energy scanning for biotin-4KC molecule with different C1-C2-C3-C4 dihedrals,
θ, obtained by both force field methods and DFT calculations.
Table 3.1 - Parameters for the surface models used in the simulations.
Figure 3.5 – Picture of the polycarbonate perfusion chamber. The labels correspond, from left
to right, to the openings for the buffer perfusion inside the chamber, the microscope objective
and the buffer flowing out of the chamber.
Chapter 4
Figure 4.1- Chemical structures of the oligopeptides (biotin-2KC, biotin-4KC and biotin-6KC) and
tri(ethylene glycol)-terminated thiols used to form the mixed SAMs tested in this research
project.
Figure 4.2 – Schematic representation of the biotin-4KC:TEGT mixed SAMs in the bio-active
state (+0.3V), where the biotin moiety is fully exposed on the gold surface, and in the bio-
inactive state, where the biotin moiety is concealed and hindered from the binding to
neutravidin.
Table 4.1 – XPS data of biotin-4KC:TEGT 1:10 solution ratio and average number of TEGT
molecules per biotin oligopeptide on the surface.
Figure 4.3 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:0 solution ratio
Figure 4.4 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:1 solution ratio
Figure 4.5 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:10 solution ratio
Figure 4.6 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:40 solution ratio
Figure 4.7 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:100 solution ratio
Figure 4.8 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:500 solution ratio
Table 4.2 – Biotin-4KC:TEGT solution ratios and respective surface ratios calculated after XPS
analysis.
Figure 4.9 – SPR sensorgram traces showing the binding of neutravidin to the biotin-4KC:TEGT
mixed SAMs at solution ratios of 1:0, 1:1,1:10, 1:40, 1:100 and 1:500 under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
Table 4.3 – Biotin-4KC:TEGT Binding Capacity (BC), expressed in Resonance Units (RU) and
Switching Efficiency calculated from SPR experiments.
Figure 4.10 – Binding capacity and switching efficiency in ON (+0.3 V), OC (no potential applied)
and OFF (-0.4 V) states of biotin-4KC:TEGT mixed SAMs at the different solution ratios tested
in this study (1:0, 1:1, 1:10, 1:40, 1:100 and 1:500).
Figure 4.11 – XPS spectra of N 1s (left) and S 2p (right) for biotin-2KC:TEGT 1:40 solution ratio
Figure 4.12 – XPS spectra of N 1s (left) and S 2p (right) for biotin-2KC:TEGT 1:100 solution ratio
Figure 4.13 – XPS spectra of N 1s (left) and S 2p (right) for biotin-6KC:TEGT 1:40 solution ratio
Figure 4.14 – XPS spectra of N 1s (left) and S 2p (right) for biotin-6KC:TEGT 1:2000 solution ratio
Table 4.4 – Solution ratios of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs and relative
surface ratios calculated by XPS.
Figure 4.15 – SPR sensorgram traces showing the binding of neutravidin to the biotin-2KC:TEGT
mixed SAMs (solution ratios of 1:40 and 1:100) and biotin-6KC:TEGT mixed SAMs (solution
ratios of 1:40 and 1:2000) under open circuit conditions, an applied positive (+0.3 V) and
negative(−0.4 V) potential.
Figure 4.16 – The surface models used in the MD simulations. The different colours in the
biotin-nKC chain represent the biotin moiety (purple), the four lysine (blue) and the cysteine
residues (dark green), respectively. Water molecules are represented by the orange dots. The
green and yellow balls denote the chloride ions and the gold atoms respectively, and the short
grey chains represent TEGT molecules.
Figure 4.17 – Conformational changes of biotin-2KC:TEGT (surface ratio of 1:8) and biotin-
4KC:TEGT (surface ratio of 1:16) mixed SAMs, under different electric field, along with the MD
simulation snapshots. The direction of the applied electric field is indicated by the black arrows.
Water molecules and hydrogen atoms are omitted for clarity. The gap distance variation
between the biotin moiety and the TEGT matrix is indicated by d.
Figure 4.18 – Conformational changes of biotin-6KC:TEGT (surface ratio of 1:15) mixed SAMs,
under different electric field, along with the MD simulation snapshots. The direction of the
applied electric field is indicated by the black arrows. Water molecules and hydrogen atoms
are omitted for clarity.
Figure 4.19 – Conformational changes of the pure biotin-4KC SAM under different electric fields
(left) and MD simulation snapshots (right). L represents the variation of the gap distance
between the biotin end group and the gold substrate.
Figure 4.1 - Molecular structures and related cartoons of the oligopeptide (progesterone-C7-
4KC) and the hexaethylene glycol-terminated thiol (EG6OH) used in the mixed SAMs, and their
calculated molecular lengths in fully extended conformations.
Chapter 5
Figure 5.2 - Cartoon representation of the ON-OFF switching system that controls the
biomolecular interaction between progesterone (red) on the surface and antibody (green) in
solution.
Figure 5.3 – XPS spectra of the a) S 2p, b) N 1s, c) C 1s and d) O 1s regions for the 1:10
progesterone-C7-4KC:EG6OH mixed SAM solution ratio.
Figure 5.4 – XPS spectra of the a) S 2p, b) N 1s, c) C 1s and d) O 1s regions for the 1:40
progesterone-C7-4KC:EG6OH mixed SAM solution ratio.
Figure 5.5 – XPS spectra of the a) S 2p, b) N 1s, c) C 1s and d) O 1s regions for the 1:100
progesterone-C7-4KC:EG6OH mixed SAM solution ratio.
Table 5.1 - Advancing and receding water contact angle and ellipsometric thickness for the
different monolayers formed for 24h. The calculated molecular lengths were determined using
ChemBio3D Ultra 12.0 in which the molecules were in fully extended conformation. The
averages and standard errors were calculated from at least three different measurements.
Figure 5.6 - SPR sensorgram, recorded in OC (no potential applied) conditions, for the injection
of anti-mouse progesterone antibody on both biotin-4KC:EG6OH and EG6OH SAMs.
Figure 5.7 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at a) 1:10, b) 1:40 and c) 1:100 solution ratio, in PBS, under OC
(no applied potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
Table 5.2 – Progesterone-C7-4KC:EG6OH Binding Capacity (BC), expressed in Resonance Units
(RU) and Switching Efficiency calculated from SPR experiments.
Figure 5.8 – Bar Chart representing the switching efficiency obtained for 1:10, 1:40 and 1:100
progesterone-C7-4KC:EG6OH mixed SAMs solution ratios
Figure 5.9 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at 1:40 solution ratio, in sEBSS + 0.3% BSA, under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
Figure 5.10 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at 1:40 solution ratio, in sEBSS + 0.1% BSA, under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
Figure 5.11 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at 1:40 solution ratio, in sEBSS + 0% BSA, under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
Table 5.3 – SPR data and switching efficiency of the progesterone-C7-4KC:EG6OH mixed SAMs
in PBS, sEBSS + 0% BSA, sEBSS + 0.1% BSA and sEBSS + 0.3% BSA respectively
Figure 5.12 – Bar chart reporting the switching efficiency of the progesterone-C7-4KC:EG6OH
mixed SAMs in PBS, sEBSS + 0% BSA, sEBSS + 0.1% BSA and sEBSS + 0.3% BSA respectively.
Chapter 6
Figure 6.1 – Steps involved in the preparation of double biofunctionalized chips. (a) Polysilicon–
chromium–gold chip, (b) Polysilicon surfaces activation by piranha cleaning, (c)
Mercaptoundecanoate-NHS SAM formation on gold, (d) TR-WGA immobilization via amide
bond formation, (e) TESUD SAM formation on polysilicon substrates and (f) F-ConA
immobilization via amide bond formation.
Figure 6.2 – Chemical structure of monomer units composing poly-D-lysine (PDL)
Figure 6.3 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions of pure 0.5 mg/ml
PDL layers on glass substrates.
Figure 6.4 – Schematic representation of silane-PDL layers on glass substrates
Figure 6.5 – XPS spectra of the a) C 1s, b) Si 2p, c) N 1s and d) O 1s regions of silane-PDL layers
on glass substrates.
Table 6.1 - Advancing and receding water contact angle and ellipsometric thickness for the
different layers formed for 24h. The theoretical molecular lengths were calculated using
ChemBio3D Ultra 12.0 in which the molecules were in fully extended conformation. The
averages and standard errors were calculated from at least three different measurements.
Figure 6.6 – Schematic representation of the imaging chamber mounted on the microscope
slide and connected to the perfusion system.
Figure 6.7 – Fluorescence images of cell adhered on PDL coated-surfaces (left), silane-PDL
layers formed using a PDL concentration of 0.1 mg/ml (centre) and silane-PDL layers formed
using a PDL concentration of 0.5 mg/ml (right).
Figure 6.8 – Fluorescence images of cell adhered on PDL coated-surfaces rinsed with ethanol
(left), and silane-PDL layers formed using a PDL concentration of 0.5 mg/ml rinsed with ethanol
(right).
Figure 6.9 - XPS spectra of the a) C 1s, b) N 1s, c) O, d) S 2s and e) Si 2p regions recorded on
clean glass substrates.
Figure 6.10 - XPS spectra of the a) C 1s, b) N 1s, c) O and d) S 2p regions recorded on clean gold
substrates.
Figure 6.11 – XPS spectra of the a) S 2p, b) C 1s, c) O 1s and d) N 1s regions of
11-mercapto-1-undecanol SAMs on gold.
Figure 6.12 – Molecular structure of 11-mercapto-1-undecanol
Figure 6.13 – XPS spectra of the a) C 1s, b) Si 2p, c) N 1s, d) S 2s and e) O 1s regions of
11-mercapto-1-undecanol SAMs on glass.
Figure 6.14 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s, d) S 2p and e) Si 2s regions of silane-
PDL SAMs on gold after the first step.
Figure 6.15 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions of silane-PDL SAMs
on glass after the first step.
Figure 6.16 - Cyclic voltammetry of bare gold after piranha cleaning (orange line), after MUD
incubation (black line) and after 5, 10 and 20 minutes of -1.5 V chronoamperometry (green,
light blue and purple lines)
Figure 6.17 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s, d) Si 2s and e) S 2p regions after thiol
removal on gold substrates.
Figure 6.18 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions incubation of glass
substrates in KOH for ten minutes, demonstrating that removal step will not affect the integrity
of the silane-PDL layer on the glass substrate.
Figure 6.19 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s, d) Si 2p and e) S 2s regions after the
formation of Progesterone-C7-4KC:EG6OH mixed SAMs on gold substrates.
Figure 6.20 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions after the formation
of Progesterone-C7-4KC:EG6OH mixed SAMs on glass substrates.
Figure 6.21 – XPS spectrum of Si 2s. A small peak is visible in the region of S 2p
A deeper insight into the molecular coverage of the glass substrates can be obtained by
calculating the ratios between the elements analysed by XPS.
Figure 6.22 – Fluorescence images of cell adhered on glass slides after the completion of the
Progesterone-C7-4KC:EG6OH mixed SAM deposition step.
Chapter 7
Figure 7.1 – Cartoon representation of double-armed switching molecule. The aspartic acid
oligopeptide arms (green) are connected to the alkyl chain (black) carrying the progesterone
moiety (red) through a core central molecule (blue) in a dendron-like structure.
List of Definitions and Abbreviations
Ab: antibody
Ag: antigen
BSA: bovine serum albumin
CV: cyclic voltammetry
CVD: chemical vapour deposition
EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EG6OH: 11-(Mercaptoundecyl)hexa(ethylene glycol)
ICSI: intracytoplasmic sperm injection
IVF: in-vitro fertilisation
MD: molecular dynamics
MUD: 11-mercaptoundecan-1-ol
NHS: N-Hydroxysuccinimide
OEG: oligo(ethylene) glycol
PBS: phosphate-buffered saline
PDL: poly-D-lysine
SAM: self-assembled monolayer
SCE: standard calomel electrode
Silane: carboxyethylsilanetriol
sEBSS: modified Earle’s balanced salt solution
SPR: Surface Plasmon Resonance
SPW: surface plasma wave
TEA: triethylamine
TEGT: triethylene glycol thiol
XPS: X-ray photoelectron spectroscopy
CHAPTER 1 1
Chapter 1 – An introduction to Bionanotechnology and Self-Assembled
Monolayers
Part of this chapter is reproduced from the paper “Electrically Responsive Surfaces:
Experimental and Theoretical Investigations”, Cantini, E.; Wang, X.; Koelsch, P.; Preece, J. A.;
Ma, J.; Mendes, P. M. Accounts of Chemical Research 2016, 49, 1223.
Abstract: This chapter provides a background to bionanotechnology, top-down and bottom-up
approaches, followed by a brief introduction to Self-Assembled Monolayers (SAMs), specific and
non-specific protein binding on surfaces and the state of the art of stimuli-responsive surfaces
for biomedical applications.
1.1 Bionanotechnology
In recent years, researchers made big efforts to develop nanomaterials and
nanostructured surfaces that can have an impact in biological and biomedical applications.
These efforts resulted in a new branch of science, called bionanotechnology. One of the aims
of bionanotechnology is to create systems and biomaterials able to mimic cellular environment
at the nanoscale, to study and understand the biological processes at cellular level. The
comprehension of cell behaviour, signalling and biological processes, can lead to the creation
of new tools that are applicable in biosensors and diagnostic, tissue engineering, regenerative
medicine and drug delivery.1,2
1.1.1 Surface Functionalisation for biosensors, cell sensing and diagnostic
Artificial biological surfaces are nowadays widely used in biotechnology and medicine
as biosensors to detect various diseases and health conditions.3,4 These surfaces are
CHAPTER 1 2
functionalised with bioactive molecules that can mimic natural processes5. The ability to
reproduce biological systems with nano-scale structures, has enabled researchers to develop
fast, low-cost biosensors, able to identify several types of biomarkers in real-time to design
efficient point-of-care (POC) clinical devices6–10.
During the last few decades, a huge research effort has been dedicated to the study of
different functionalisation strategies for the development of novel, simple, low-costs, portable
sensing devices applicable to biomedical diagnostic3,10, such as devices for diagnosis of
infectious diseases11, cancer biomarker detection12–14 metabolite quantification in living cells15,
DNA biosensing16,17 and identification of antigen-antibody interactions18,19.
Biosensing platforms can be obtained by chemically modifying a vast range of
surfaces5,16 such as flat metal surfaces20, nanoparticles21–24 and nanotubes25,26, in a tailored
manner with the desired chemistry and surface morphology21,27. In particular, carbon
nanotubes (CNTs) are widely used in current biomedical research, thanks to their mechanical,
electrical, magnetic and optical properties that make them suitable tools to create devices with
several biomedical applications, from drug delivery28–30 to diagnostics27,31.
Various surface functionalisation strategies are available, such as layer-by-layer (LbL)
strategies32–34 and Self-Assembled Monolayers (SAMs) formation35–38.
Layer-by-layer functionalisation of surfaces comprises several steps of layers addition
or modification, to obtain the desired layered structures. Specifically, Sung et al.39 used LbL-
coating to modify polydimethylsiloxane surfaces to decrease the amount of non-specific
binding and improve the detection of low levels of proteins. On the other hand, the formation
of SAMs consists of a single step, where one or more component molecules organise
themselves in an ordered nanometric monolayer on the substrate. SAMs will be described in
detail in section 1.3.
CHAPTER 1 3
1.1.2 Other materials used in biosensing
In addition to metal surfaces, metal nanoparticles and nanotubes mentioned, other
materials, such as nanoporous materials40–42, nanozymes43,44 and hydrogels45 have gained
increasing interest for biomedical research.
Nanoporous materials40,41 possess a bigger surface-to-volume ratio than conventional
nanomaterials, offering an enhanced signal when the analyte interacts with the surface. In
addition, they can be functionalised to mimic the protein nanochannels found on cell
membranes. Therefore, they can be of paramount importance in the study of nanochannels
single-molecule sensitivity and selectivity42 . By tailoring the size of the nanopores, it is possible
to obtain a selective control over the molecular transport through the nanochannels. These
powerful materials can be made by self-ordering synthesis based on electrochemical
anodization, and both anodic aluminium oxide (AAO) and titania nanotubes (TNTs) have
already been used in biosensors fabrication46,47 . In addition to the increased surface areas,
such nanoporous materials can be prepared at relatively low costs and show chemical
resistance, thermal stability, hardness and biocompatibility. These characteristics make them
very useful when developing ultra-sensitive biosensing devices40–42.
Nanozymes8,43 are artificial enzymes presenting enzyme-like activities and are widely
used in biomimetic, which attempt to mimic the characteristics and functions of natural
enzymes. Some of the materials that have shown unexpected enzyme-like characteristics43 are
fullerenes48,49, metal nanowires50,51, nanorods52 and several types of metal nanoparticles48,53.
Such materials possess both the characteristics of natural enzymes and the properties of the
material used to fabricate them.
For example, Ali et al.48 were able to create a tris-malonic acid derivative of fullerene
C60 molecules, capable of transforming the dangerous superoxide radical (O2-) into oxygen
CHAPTER 1 4
molecules (O2), mimicking superoxide dismutase (SOD) enzymatic activity. Wang et al.48 have
demonstrated the use of nanoparticles, chemically modified with oligonucleotides molecules,
able to induce RNA cleavage and applicable to the treatment of Hepatitis C.
Researchers have been capable of mimicking other natural enzymes, such as
catalases53, oxidases54 and peroxidases55,56 making nanozymes unique, and applicable in
sensing, imaging and therapeutics.
Hydrogels are hydrophilic materials formed by a polymer network that can absorb
water from 10% to thousands times their dry weight57 . By changing their chemical and physical
characteristics, their structure can be tailored, and they can be integrated into micro-systems.
Their biocompatibility and their sensitiveness to external stimuli make them largely employable
as switchable biosensors to detect changes, for instance, in pH58, monitoring biological
processes57, DNA sensing58, carbohydrate sensing59 and toxin screening59. Switchable
materials will be described in detail in section 1.5.
1.2 Functionalisation approaches
This section describes the two approaches used as surface functionalisation strategies,
namely the top-down and the bottom-up approach60 (Figure 1.1).
CHAPTER 1 5
Figure 1.1 – Cartoon representation of top-down and bottom-up approach
1.2.1 Top-down approach
This approach consists in the removal of matter from a bulk material to achieve the
desired smaller structure showing a specific order and shape60,61. The most used technique in
this method is nanolithography that allows the construction of a pattern on the
surface/material. There are many lithographic techniques that can be exploited, classical
methods such as electron-beam lithography and photolithography or novel methods, such as
dip-pen lithography62, nanoimprint lithography63,64, colloidal65 and soft lithography66,67. In the
classical methods, a substrate, covered in a polymer layer (e.g. resist) is irradiated with either
UV light or an electron beam, to carve the resist layer and create a nanometric pattern (e.g.
nanopattern). Then, using a technique called “etching”, the nanopattern is transferred to the
substrate surface68,69 (Figure 1.2).
CHAPTER 1 6
Figure 1.2 – Schematic representation of the photolithographic process. The substrate covered
with a masking film (A) is coated with a photoresist (B). A mask is then collocated on the
photoresist film (C) to create the desired pattern after UV exposure (D) and etching (E). Finally,
the photoresist is completely removed by stripping, to obtain the desired patterned substrate
(F).
The novel methods have been developed to overcome the constraints of common
lithography. Soft lithography, for example, is a low-cost and flexible technique extensively used
due to the possibility of creating large-scale nanostructured architectures, simply using a mold
or a mask made using a nanopatterned elastomer70,71. The disadvantage of this technique is
the need of being supported by standard lithography methods to create the molds and the
masks. To solve this problem colloidal lithography has started to be extensively used72. This
CHAPTER 1 7
technique uses nanodisperse colloidal particles which size goes from around 10 nm to around
10 m that can self-assemble in 2D- and 3D-periodic arrays called colloidal crystals to create
masks on the substrate surface67,73. The application of this kind of lithography is convenient
when there is the necessity for nanostructures presenting periodic arrangements65,73.
1.2.2 Bottom-up approach
The bottom-up methods, extensively used and studied as surface functionalisation
methods60, are based on the spontaneous self-assembly of small molecules into two-
dimensional and three-dimensional composite structures showing defined physical and
chemical properties, created atom-by-atom through covalent, non-covalent, ionic and metallic
bonds and weak interactions, such as dipole-dipole and Van der Waals interactions74,75.
The most broadly used nanofabrication method in this approach are Langmuir-Blodgett
(LB)76,77 and Self-Assembled Monolayers (SAMs)78–81. These methods allow the creation of well-
ordered and packed monolayers by using a wide range of different molecules. However, the
Langmuir-Blodgett approach presents the disadvantage of requiring long construction times,
molecules with specific characteristic (i.e. amphiphilic) and expensive instrumentation. In
addition, LB layers present limited mechanical stability and robustness because the fabrication
does not involve chemisorption between the molecules and the substrates82. The lack of strong
molecular interactions limits the applicability of LB layers in ambient and physiological
conditions, limiting their suitability for biological and biomedical applications82.
On the other hand, SAMs are commonly used in bionanotechnology due to their ease
of fabrication and the possibility of tailoring their properties depending on desired purpose
that overcome the drawbacks presented by some types of SAMs (e.g. thiol SAMs) such as
CHAPTER 1 8
limited stability and robustness78,83. The next section describes in detail the characteristics and
the possible uses of SAMs.
1.3 Self-Assembled Monolayers
SAMs have been intensively studied for the last decades. The first publication on the
preparation of a molecular layer by adsorption is dated 194684, but the potential of SAMs was
published only when Nuzzo and Dallara demonstrated that SAMs of alkanethiols can be
prepared by adsorbing n-alkyl disulphides on gold substrates from diluted solutions85. SAMs
were then thoroughly studied to tailor surfaces with various properties, on different substrates
such as gold86–88, indium tin oxide (ITO)89, glass90,91, silicon wafers (SiO2)92,93and nanoparticles94.
By carefully choosing both the head and the end groups of the molecules composing the SAMs,
the characteristic of the surfaces, such as hydrophilicity/hydrophobicity charge and the type of
biomolecules composing the end groups can be changed to obtain a monolayer that can be
used in a wide range of applications, in both engineering (e.g. sensors95, coating
technologies96,97, optics95 and informatics95) and biomedical (e.g. biosensors98, tissue
engineering99,100 and point of care diagnostics101,102) fields.
1.3.1 Surfactant
During the process of self-assembly, surfactant molecules adsorb on a solid surface
(substrate) to form well-ordered molecular assemblies78,87. These molecules are formed by
three parts: the head group, the backbone and the end group (Figure 1.3).
CHAPTER 1 9
Figure 1.3 – Schematic representation of a surfactant molecule
The head group binds to the substrate surface, and therefore its choice depends on the
substrate material used. The most commonly used SAMs are thiol molecules absorbed on gold
or silver via the sulphur headgroup. Other head groups include silanes for SAMs on silicon
oxide, silicone, mica and glass or carboxylic acid groups in the case of AgO/Ag substrates86,87,103–
107.
The backbone represents the central part of a surfactant molecule. It represents the
connector between the head- and the end group, but it also plays an important role in the
chemisorption process, depending on its characteristic78,83,87,106–108. By changing the length of
the chain composing the backbone, it is possible to control density, orientation and ordering of
SAMs molecules on the surface, e.g. long-chain alkanethiols (HS(CH2)nX, n>10) form densely-
packed and well-ordered SAMs, at a 30˚ tilt angle on the surface79,80,103.
CHAPTER 1 10
Finally, by varying the end group, the SAM wettability, charge, together with the type
of biomolecular interactions occurring on the surface can be tailored, allowing a wide range of
applications in both engineering and biomedical fields80,87.
1.3.2 Substrates and SAMs
Different substrates materials and surfactant molecules can be used in the preparation
of SAMs. The following sections describe the SAMs used in this research work: silane SAMs and
thiol-based SAMs.
1.3.2.1 Silane SAMs
Silane SAMs were first introduced by Sagiv in 1980, that studied the adsorption of
n-octadecyltrichlorosilane (OTS) on glass substrates109. Many studies were conducted on silane
monolayers in the past decades due to their potential many applications in a wide range of
research areas, such as thin film technology, micro- and optoelectronics, chemical sensors and
protective coatings. Silanes can also be used in biosensors, as bioactive surfaces or for cell
adhesion and protein adsorption98,110,111. Silanes self-assemble via an hydrolisation reaction of
the headgroup (trichloro-, trimethoxy- or triethoxysilane) that subsequently react with the
hydroxyl groups (-OH) on the substrate surfaces (Figure 1.4), forming a cross-linked network of
Si-O-Si bonds covalently linked to the substrate itself93,112 .
CHAPTER 1 11
Figure 1.4 – Cartoon representation of a silane SAM.
Several research groups reported the possibility of the formation of islands of silanes
on the surfaces, not creating a homogeneous layer112,113. This process strongly depends on
several parameters, such as temperature, solvent used, pH, water content, and age of the
solution. These parameters have to be carefully controlled, e.g. trichlorosilanes are sensitive to
water and their headgroup present high reactivity, reducing the number of possible endgroups
that can be incorporated into the surfactant molecule104,111. However, the water cannot be
completely removed because its absence leads to an incomplete monolayers
formation111,112,114. These problems can be circumvented by creating the desired silane layer in
more than one step: 1) molecules able to give a specific surface organisation are used to create
a precursor SAM and 2) the precursor SAM is modified via chemical surface reactions to obtain
a well-organised silane layer111,115. Despite the challenges illustrated, silane monolayers are
widely studied and used due to their greater thermal and mechanical stability compared to that
of thiols on gold104,105,116. In addition, such monolayers prepared on smooth surfaces like silicon
CHAPTER 1 12
wafers, present remarkable properties such ultra-low surface roughness, controlled wettability
and chemical homogeneity that can be regulated by varying the silanes endgroup104,117.
1.3.2.2 Thiol SAMs
SAM surfaces formed by adsorption of thiol molecules can be prepared on different
metal substrates such as gold, silver, platinum, and copper.83,94 However, thiol SAMs on gold
are the most commonly used and studied due to their ease of preparation and characterisation.
In addition, they play an important role in the building of a wide range of devices and systems
that can have important applications in nanotechnology100. Alkanethiols SAMs with chain
length between 10 and 22 carbon atoms have been extensively analysed79,80 and it is known
that alkyl chains self-assemble rapidly and spontaneously in a well-ordered and oriented
manner, following a three step process (Figure 1.5)83,94,100.
Figure 1.5 – Schematic of the three-steps process for SAMs formation. 1) Physisorption, 2)
chemisorption and 3) completion of SAM spatial orientation and packing.
CHAPTER 1 13
The first step is a physisorption process that depends on thiol concentration. The time
necessary for this process increases as the thiol molecules concentration decreases. Thiol
molecules lay parallel to the surface for all the duration of this step118. The second step is a
chemisorption process during which the surfactant chains go from a disordered state to a more
ordered one, organising themselves in a two-dimensional crystal118. The kinetics of this step is
influenced by both the head group-substrate interactions and the chain-chain interactions in
the molecules of the substrates (Van der Waals, dipole-dipole)83,94,100. This step takes hours and
it is faster if the alkyl chains are longer78. The last step is the completion of the orientation and
packing of surfactant molecules on the surface to obtain a well-ordered and stable SAM. This
process is usually completed in 24 h. However, this has been demonstrated only for alkyl thiols
with a backbone length >10 carbons84,85. If the backbone chain is shorter, the thiol molecules
cannot reach a correct two-dimensional crystal structure during the second step and this leads
to a disordered arrangement of molecules on the substrate surface that is not uniformly
covered. Various electron diffraction studies, performed in the late 80s and early 90s showed
that alkanethiolates adopt a (√3 x √3)R30˚ structure on Au (111) substrates119. In such structure,
sulphur atoms are organised in an hexagonal arrangement, separated by a distance of
4.97Å78,119. Such studies suggested that each sulphur atom is bound to the 3-fold hollows
present on the gold lattice, in a highly-ordered manner120.
In addition, each alkanethiol molecule has an area of 2.14Å2 and a cross-sectional area
of 2.14Å2. This difference forces the alkyl chains to tilt by an angle of around 30˚ to the gold
surface normal (Figure 1.6)
CHAPTER 1 14
Figure 1.6 – Schematic representation of the arrangement of dodecanethiol molecules on Au
(111) substrates to achieve maximum surface coverage. (a) Structural model of the hexagonal
arrangement of thiols (black hexagon) on gold and the area covered by each thiol molecule
(dashed-lined circles). (b) Cross−section of the SAM formed showing the alkane chains 30˚
tilting in the direction of their next−nearest neighbours.
This tilting angle allows the alkyl chains to maximise Van der Waals chain-chain
interactions, leading to highly-packed monolayers78,119.
1.3.3 Mixed SAMs
The preparation of mixed SAMs (multi-component SAMs) is more challenging than
forming a single-component SAM. The routes that can be followed are usually two: 1)
modifying the end groups of a single-component SAM in a selective manner, using different
techniques such as photolithography92, electron-beam lithography68 and micro-contact
printing121,122 or 2) combination and co-adsorption of two or more different surfactants onto a
surface87,123 (Figure 1.7). The second method of preparation is the most widely used one.
CHAPTER 1 15
However, the composition of the mixed SAM on the surface can differ from the one of the
mixture of surfactants in solution.
Figure 1.7 – Cartoon representation of a mixed SAM
This is due to backbone interactions between the molecules composing the mixed
monolayer, but also to solvent-surfactant interactions that lead to the preferential adsorption
of one of the components onto the surface83,87,123,124. In addition, the phase separation
phenomenon can limit the formation of homogeneously-ordered mixed SAMs119,125–128. This
event does not allow the mixed SAM components to bind to the surface showing a specific
distribution119,129. The gold-thiolate interface can be considered motile, as demonstrated by
several studies130,131. These studies showed that thiol molecules can diffuse on gold surfaces
and exchange their position with another thiol. In particular, mixed alkanethiol SAMs can
arrange into discrete domains at a diffusion rate independent from the alkanethiol chain
length132. By carefully tailoring the composition of the mixed SAM, it is possible to overcome
these limitations and control the exposure of active molecules on the surface, which can be
used to control several biological and chemical interactions (e.g. separation of molecules133,
CHAPTER 1 16
specific biomolecular interaction134) and fabricate sensitive biosensors (e.g.
immunosensors135).
1.3.3.1 Unspecific protein adsorption
As already stated in the previous sections, SAMs can be exploited to build sensitive
molecular recognition systems. To obtain devices with optimum performances, the non-
specific adsorption of unwanted molecules onto the substrate surface has to be reduced. These
undesired molecules can hinder or block the binding sites on the active molecules composing
the monolayer, reducing the efficiency of the biorecognition system. Non-specific binding can
either increase the background noise, or give a “false positive” response136. Mixed SAMs are
particularly appropriated for this purpose. By selecting the chemical functions on the surfactant
molecules, it is possible to decrease the amount of unspecific binding. Several studies have
been conducted on the use of oligo(ethylene glycol) (OEG)-terminated SAMs to suppress
protein unspecific binding136–138. Prime and Whitesides, in 1993137, studied the adsorption of
four proteins on different SAMs on gold. Alkane-thiols, alkyl-alcohol thiols and oligo(ethylene
glycol)-thiols were investigated. The latter monolayer was the only one able to resist the
adsorption of all the four proteins tested. OEGs can therefore be used in numerous biological
applications where unspecific protein adsorption has to be prevented134,136. The ability of OEGs
SAMs to create a high surface coverage, make them able to resist non-specific protein
adsorption.137,139 These molecules are therefore often used as one of the surfactants
composing multicomponent SAMs. By varying the ratio of OEGs molecules and bioactive
molecules, it is possible to control the spatial distribution. This spatial control allows the best
interaction between the bioactive surfactant and the target molecules of interest. In addition,
CHAPTER 1 17
the length of OEGs chains and the number of ethylene glycol units affect the capability of OEGs
SAM to block protein adsorption. OEG alkanethiols monolayers formed by (CH2)11-(OCH2CH3)n-
OH (EGn) chains, where n<3, are inefficient in suppressing protein adsorption140,141. The
protein-repellent characteristics are due to the presence of strong repulsive forces between
OEGs SAMs and proteins. Thanks to the presence of several oxygen atoms in the ethylene glycol
units, these monolayers can form hydrogen bonds with water molecules. This interaction
creates a hydrophilic layer that repels proteins as a result of the repulsive hydration forces
action141,142. However, the presence of ethylene glycol thiol molecules does not have to block
specific protein binding with bioactive molecules when needed.
1.3.3.2 Specific protein adsorption
In the case of protein affinity studies on surfaces, the receptor molecules composing
the mixed SAM must interact in a specific manner with the protein of interest. Theoretically
any protein-receptor system can be studied on such mixed architectures, but the most widely
explored are the avidin-family proteins binding to biotin (receptor). This system has been used
for several years in various application in biotechnology143, such as immunoassays143–147,
biosensors148, tissue engineering149,150 and drug delivery151,152. All these systems exploit the high
affinity and specificity between biotin and the avidin-family proteins. However, avidin also
presents high non-specific binding153, therefore streptavidin154–156 and Neutravidin157–159 are
preferred. Avidin-family proteins are tetramers with a mass between 50 and 70 kDa, composed
of four identical subunits, exhibiting extremely high binding affinity to biotin, with dissociation
constants KD on the order of ≈10-14-10-15 mol/L and forming a stable complex at different
temperatures and pH values153 (Figure 1.8).
CHAPTER 1 18
Figure 1.8 – Cartoon representation of biotin binding to two of the four binding sites of the
avidin family protein
Avidin is a positively charged protein, that can interact with negatively charged
substrates, such as silica based substrates159 or cells membranes153. The isoelectric point of
avidin is unusually high153 (pI≈10) and the presence of carbohydrate moieties, formed by four
mannose and three N-acetylglucosamine molecules per subunit, may explain why avidin is
prone to high non-specific binding160. Neutravidin is a deglycosylated form of avidin, with a
lower isoelectric point (pI≈6.3), whereas streptavidin is a non-glycosylated protein with a more
acidic isoelectric point (pI≈5.6)153.
Another type of specific binding widely studied in biotechnology is the antigen-antibody
interaction161,162. Antibody-based biosensors163 give the possibility of a rapid and sensitive
detection of heavy metal ions164, proteins blood levels165–167, allergens168 and pathogens163,169.
Immunosensing exploit the immune system of a host (i.e. murine, leporine, ovine or avian), by
injecting the molecules or cells of interest into the host’s body, triggering an immune response.
The immunoglobulins (IgG) produced by the host animal are collected after several
immunisation processes. They present a structure composed by two heavy chains and two light
chains170 (Figure 1.9).
CHAPTER 1 19
Figure 1.9 – Schematic representation of an antigen-antibody system. Several antigens are
available for binding, but only one is specific for the antigen binding site on the antibody
chains
Different types of antibodies can be produced: polyclonal, monoclonal and
recombinant. Polyclonal antibodies (pAb) are usually produced using rabbits, goats or sheep as
hosts171 and frequently used in sensors for pathogens detections. Polyclonal antibodies
recognise different epitopes on a single cell, therefore, when high specificity is required, the
use of monoclonal or recombinant antibodies is preferable. Monoclonal antibodies (mAb) are
produced using the hybridoma technology and mice are the most common used hosts. The
spleen, together with the bone marrow and primary lymph nodes are employed as a source of
B cells to obtain antibodies that are then fused to immortal myeloma cells171. The resulting
CHAPTER 1 20
hybrid cells (hybridomas) secrete antibodies that recognise a single epitope on a single cell.
Recombinant antibodies are prepared by using phage-display technology and antibodies
libraries against the target of interest to achieve the detection of numerous antigens, including
proteins, haptens and carbohydrate moieties171.
Both biotin-neutravidin and antigen-antibody systems will be developed in this research
work and will be described in detail in Chapter 3 and Chapter 4.
1.3.4 Dynamic SAMs
In recent years, researchers have focussed their studies on the development of stimuli-
responsive surfaces. Such surfaces can be obtained by changing the end group and/or the
backbone of the surfactant molecules composing SAMs, to create moieties presenting
switchable/dynamic characteristics1,172–175. These tunable moieties can respond to a wide
range of stimuli, such as temperature (thermo-responsive surfaces)176–182 , light (photo-
responsive surfaces)183–185, pH and concentration (chemical/biological stimuli-responsive
surfaces)186–191, magnetic field (magneto-responsive surfaces)192,193 and electrical potential
(electrical-responsive surfaces)10,194–199. Smart surfaces have gained paramount importance
due to the possibility to obtain a high spatial and temporal control, a rapid response and to be
used in a wide range of biological and biomedical applications. To date, switchable surfaces
have been employed in drug delivery179,181, to control specific and unspecific protein
binding196,197 and mammalian and bacterial cell attachment and detachment10,182,195,198–200, but
also in tissue engineering201,202 and regenerative medicine200,203. The application of an external
stimulus results in a switch that can be either chemical or conformational (Figure 1.10).
CHAPTER 1 21
Figure 1.10 – Schematic of the possible switches that can be obtained upon the application of
a stimulus. a) Chemical switch: the chemical composition of the monolayer varies when an
external stimulus is applied (e.g. a change in pH). b) Conformational switch: the chemical
composition of the monolayer remains the same, while its conformation varies when an
external stimulus is applied (e.g. application of an electrical potential).
In the case of conformational switching, a high control of the monolayer spatial order
must be achieved. Conventional SAMs (i.e. alkanethiols SAMs) are too high-density to allow
conformational changes, therefore the switching is hampered204. Each molecule must possess
enough spatial freedom, as shown by Lahann and co-workers in their study205. They designed
a surface which wettability can be dynamically changed. Firstly, a low-density
16-Mercaptohexadecanoic acid (MHA) SAM was created on a gold surface. MHA molecules
CHAPTER 1 22
create well-ordered and highly-packed SAM on gold, presenting both a hydrophobic alkyl chain
and a hydrophilic carboxylate end group. Therefore, to give the monolayer enough space on
the surface to undergo a conformational switch, a globular end group (spacer) was linked to
the carboxylate moieties, prior of MHA SAM formation. The result is a monolayer with a low-
density molecular distribution on the substrate. After the removal of the globular spacer by
hydrolysis, a negatively-charged SAM is obtained that can then be exploited to induce a
dynamic conformational change of MHA molecules. Upon the application of a positive electrical
potential, the negative carboxylate groups are attracted towards the gold surface. This
molecular change causes the exposure of hydrophobic alkyl chain, changing the wettability and
making the surface hydrophobic overall. The process can be reversed by simply applying a
negative electrical potential, causing the repulsion of the carboxylate end groups from the gold
surface and creating a hydrophilic surface.
Another important research work was conducted by Liu and co-workers206 starting from
the findings of Lahann’s group205. A low-density SAM (LD-SAM) was created on gold surfaces,
by assembling MHA molecules pre-capped with cyclodextrins (CD) of different dimensions,
covalently linked to the carboxylic end groups. The LD-SAMs created after removing the CD-
caps, where used to selectively control the attachment of two proteins (avidin and streptavidin)
in a reversible manner over the surface, by applying an electrical potential. When a negative
potential is applied, the carboxylate groups are fully exposed on the surface and the positively
charged avidin is adsorbed to the surface, whereas the negatively charged streptavidin, shows
an opposite behaviour. Streptavidin was also used to demonstrate a similar adsorption and
release process on an amino-terminated LD-SAM (Figure 1.11).
CHAPTER 1 23
Figure 1.11 – Illustration of the formation and switching of a LD-SAM by employing a
precursor molecule presenting a bulky head-group. Upon the application of an electrical
potential the monolayer can reverse its wettability by switching between hydrophilic and
hydrophobic states.
In addition, Liu and co-workers demonstrated that the application of an electrical
potential has no effect on protein adsorption for high-density (HD) SAMs, due to the steric
hindrance that hampers the bending of the alkanethiolate molecules on the surface. Starting
from the pioneering work of Lahann and Liu, several studies have been conducted on the
development of smart surfaces10,173,207–209. This thesis research work will focus only on the use
of electrically switchable surfaces, which will be described in the next section.
1.3.5 Electrically-switchable SAMs
Electrically-switchable surfaces have gained increasing interest during the past decade
since it allows control over the interaction between surfaces and peptides173, proteins206,208,
DNA210 and cells173,199. The rapid response to an electrical stimulus is fundamental for many
vital cellular signalling pathways in the body, e.g. the voltage-dependent of sodium and
CHAPTER 1 24
potassium currents across cell membranes, triggered by an electrical signal coming from the
nerves211,212 and necessary for a correct functioning of the nervous system.
Researchers have been combining the observation of natural processes with advanced
biotechnological techniques to fabricate switchable architectures that can control processes at
the micro- and nanoscopic scale and mimic complex cellular mechanisms. An interesting label-
free method to control and analyse the interactions between proteins and ligands on the
surfaces was presented by Knezevic and co-workers in 2012213. In this system, negatively
charged DNA strands (“levers”) tethered to a gold surface, were functionalised at the top end
with a cyanine 3 (Cy3) dye. When a positive potential of +0.3V was applied, it caused an
attraction of the negatively charged lever toward the substrate, causing a reduction in
fluorescence emission from the dye, caused by the quenching effect of the metal surface. On
the contrary, when a negative potential of -0.5V was applied, it caused the repulsion of the
DNA strands from the surface, exposing the Cy3 dye molecules, therefore increasing the
fluorescence emission. By measuring the fluorescence emission, it is possible to calculate the
distance between the DNA’s top end and the substrate. When the DNA lever is functionalised
with a protein at its top end, it is possible to quantify the binding kinetics (Kon, Koff rate
constants), the dissociation constant (KD in picomolar regime) and the influence of competitive
binders (EC50 values). In this case, there is a delay behind the dynamics of the bare level, due
to the presence of a hydrodynamic drag occurring when a protein is bound to the DNA’s distal
end (Figure 1.12).
CHAPTER 1 25
Figure 1.12 – Schematic representation of the DNA lever; a) lying on the surface when a
positive potential is applied and repelled when a negative potential is applied, fully exposing
the fluorescent dye. b) When a protein is linked to the DNA’s top end, the upward motion is
slowed and lags behind the bare lever. The dark yellow circle represents the Cy3 dye when the
fluorescence is quenched by the gold surface. The bright yellow circle corresponds to a high
fluorescence emission, due to the DNA molecule carrying the fluorophore being fully extended
on the surface.
In addition, this method allowed the calculation of the protein diameter with angstrom
resolution, by analysing the time-resolved upward dynamics, but also to collect information
about the avidity effect and discriminate between analytes presenting multiple binding sites.
CHAPTER 1 26
An excellent contribution to the development of electrically-switchable surfaces was
given by our group. In the first work196, switchable surfaces where created by preparing a
mixed SAM on gold surfaces, composed by oligopeptide molecules that can undergo reversible
molecular changes upon the application of an electrical potential and molecules of triethylene
glycol thiol (TEGT) acting as lateral spacers to space out the switchable backbone such that the
conformational changes are not hampered by steric limitations. The dynamic oligopeptide is
formed by a chain of four positively-charged lysines (4K) linked to a molecule of biotin at the
top end and to a cysteine (C) at the bottom end for the tethering to the gold surface. The
bioactive molecule (biotin), can be reversibly exposed (ON state) or concealed (OFF state) by
applying an electrical potential to the gold surface (Figure 1.13).
Figure 1.13 – Schematic representation of the switching of Biotin-4KC:TEGT mixed SAM upon
the application of an electrical potential.
CHAPTER 1 27
The dynamics of the molecular changes were followed in real-time using surface
plasmon resonance (SPR) and fluorescence microscopy, to study the specific binding between
biotin and fluorescently labelled Neutravidin196. When a potential of +0.3V was applied, high
Neutravidin binding was observed both by SPR and by recording an increase in fluorescence,
meaning that the biotin moiety is fully exposed on the surface and the interaction with the
protein is maximised. On the other hand, when a negative potential of -0.4V is applied, the
charged backbone collapses on the surface and the biotin moiety are hindered by the ethylene
glycol-terminated thiol molecules and the interaction with Neutravidin is minimal. The bio-
inactive state leads to more than 90% reduction in protein binding. The biotin-4KC:TEGT mixed
SAM surface was further developed in this research work and experimental studies were also
conducted on a shorter (biotin-2K) and longer (biotin-6KC) oligopeptides to study the influence
of the length of the charged backbone on switching. The findings will be illustrated in Chapter
3.
In 2013 Pranzetti et al10,195, tested the use of switchable surfaces to analyse bacterial
adhesion by forming a two-component SAM of 11-mercaptoundecanoic acid (MUA)
representing the switching unit and the backfiller mercaptoethanol (MET) that can reversibly
change its wettability upon the application of an electrical potential. By varying the potential
in cycles, between +0.25V, OC (no applied potential) and -0.25V, it was possible to follow the
interaction of the mixed SAM with the hydrophobic marine bacterium Marinobacter
hydrocarbonoclasticus (Mh) (Figure 1.14).
CHAPTER 1 28
Figure 1.14 – Cartoon representation of the control of bacteria cells adhesion by switching a
MUA:MET mixed SAM.
When a positive potential was applied, the carboxylate groups on MUA were attracted
toward the substrate, exposing the hydrophobic alkyl chains, causing the repulsion of bacteria.
It was demonstrated that the attachment of cells can be reversed by moving from a negative
potential to a positive one, but the increase of the number of ON/OFF cycles reduce the
reversibility of the process. In addition, it was shown that a pure SAM of MUA is not affected
by the change of the surface potential.
The studies described above were conducted in limited biological conditions,
phosphate buffer saline (PBS) solution was used to analyse the interaction between biotin and
Neutravidin, whereas artificial seawater was used in the case of monitoring bacterial adhesion.
Starting from the encouraging results obtained in simple media, Lashkor et al.198 investigated
whether more complex biological conditions can affect the efficiency of the induced
conformational changes. The switching of biotin-4KC mixed SAMs with various ethylene glycol-
terminated thiols was investigated, by electrochemical SPR in real-time, in different media
commonly used for cell and tissue culture, namely Dulbecco’s modified Eagle’s medium
(DMEM) containing a mixture of inorganic salts, amino acids, glucose and vitamins, DMEM
containing 10% foetal bovine serum (DMEM-FBS) and DMEM-FBS with (4-(2-hydroxylethyl)-1-
piperazineethanesulfonic acid) HEPES buffer (DMEM-FBS-HEPES). The results showed that,
CHAPTER 1 29
when the spacer is a longer ethylene glycol thiol (11-carbon TEG-terminated thiol, C11TEG),
the surface is more resistant to non-specific binding coming from the medium, but the
switching efficiency is reduced in PBS if compared to the biotin-4KC:TEGT tested before.
However, the high protein resistance, make this mixed SAM suitable for the creation of systems
that have to operate in complex biological conditions. The dynamic changes were then
monitored in the three different media indicated above. From the results, it is possible to infer
that both FBS and HEPES both interfere with the switching process, by interacting with the
oligopeptide chains on the surface, but the presence of DMEM reduces the antagonistic effect.
By diluting the media solutions, the switching efficiency increases, meaning that by carefully
controlling the complex biological conditions it is possible to exploit charged oligopeptide
chains to achieve maximum conformational changes. After having demonstrated the feasibility
of conformational transitions in complex media, the study was developed further to investigate
cell adhesion on dynamic SAMs. Putting together the findings of the previous studies, Lashkor
et al. developed an arginine-glycine-aspartate (RGD) oligopeptide-based surface199, able to
regulate cell adhesion. RGD is a tripeptide commonly present in the majority of the adhesive
proteins on the extracellular matrix and it is specific for integrin-mediated cell adhesion. The
switching unit was constituted by a 3-lysine oligopeptide (C3K) functionalised with a glycine-
arginine-glycine-aspartate-serine (GRGDS) recognition unit to obtain a C3K-GRGDS:C11TEG
mixed SAM on gold substrates. The switching performance was followed by electrochemical
SPR using DMEM as medium. When a negative potential was applied (-0.4V), the oligopeptide
switching unit was in a collapsed conformation on the surface, hampering cell adhesion. If the
conformation/orientation of the RGD peptide is altered upon the application of an electrical
potential, it is possible to regulate the exposure and availability of RGD sites for cell surfaces
receptors. However, it is not possible to perform switching cycles from adhesive- to
CHAPTER 1 30
resistant-state, as in the case of bacteria adhesion195 described before, due to the cell
attachment being directed by multiple RGD-integrin bonds in parallel.
Understanding the mechanisms of dynamic changes on the surface is of paramount
importance to have a complete insight into the molecular reorganisation that occurs when an
electrical stimulus is applied. Two fundamental techniques that can provide numerous
information about the switching process are molecular dynamics (MD) simulation and sum-
frequency generation (SFG) spectroscopy, the former have been used in this research work in
collaboration with Nanjing University (China) and will be described in Chapter 3. SFG was used
by Pranzetti et al194 to study the orientation of biotin moieties on the surface in the biotin-
4KC:TEGT mixed SAM. Such technique exploits IR overlapped to visible laser pulse to excite the
vibrational states in the molecules of interest and allows the measurement of SFG spectra. By
using a special electrochemical cell, it was possible to monitor in real-time the changes
occurring on the surface when a positive (+0.3V) and a negative (-0.4V) potential were applied
and characterise a molecular vibration associated with the biotin moiety in the mixed SAM.
When a positive potential is applied, the switching backbone is fully extended on the surface,
exposing biotin in an anisotropic upright orientation resulting in a dip of the biotin vibration
within isotropic biotin orientation and a very weak SFG peak is recorded.
1.4 Application of switchable surfaces to the selection of sperm cells for in-vitro fertilisation
(IVF) techniques
Male infertility is the male’s inability to induce pregnancy in a fertile female214 and it
affects the 7% of men215. Infertility is commonly due to abnormalities in semen quality215,216
that can be either caused by age217, lifestyle218,219, oxidative stress220, autoimmune reactions221
CHAPTER 1 31
or genetic problems222. One of the genetic factors leading to infertility in males, is the incorrect
expression of genes coding for the calcium channels CatSper. These channels are a family of
sperm-specific cation channels223, composed by four pore-forming channel proteins, CatSper
1-4, and three subunits, CatSper, CatSper and CatSper, which control the calcium ion (Ca2+)
influx, required for hyperactivation224–227. CatSper are permeable channel proteins located on
the plasma membrane of spermatozoa228,229. Blocking these channels in knockout spermatozoa
lead to an impairment of normal sperm function223,225. The opening of the calcium channels is
regulated by the interaction between sperm cells and progesterone230. Therefore, sperm cells
that are able to respond to progesterone will then acquire the ability to fertilise the female
egg231,232.
Progesterone, is a steroid hormone, released by cumulus cells surrounding the oocyte
in the female oviduct230,233. The release of progesterone activates the CatSper channel and
therefore the calcium influx into the sperm flagellum234,235. When good quality sperm cells
interact with progesterone, they undergo a change in their flagellar activity, moving from
symmetric to asymmetric232,236. If one of the genes coding for one of the CatSper subunits is
suppressed, the sperm cells are unable to start the Ca2+ influx in response to progesterone and
acquire their fertilisation state226,237. It has been demonstrated that in both human and mice,
all the four subunits composing the CatSper channel are needed for a successful control of
spermatozoa hyperactivation, chemotaxis and acrosome reaction238–240.
When spermatozoa fail to achieve fertilisation naturally, in-vitro fertilisation (IVF)
methods are necessary to help couples in conceiving241,242. Two methods are generally used:
the first one is standard IVF, where, after the stimulation of the female ovulation, an egg or
eggs (ovum or ova) are removed from the woman’s ovaries and incubated with sperm in an
CHAPTER 1 32
adequate medium, in a laboratory241,243. The fertilised eggs (zygotes) are then cultured for 2-6
days in a growth medium and then implanted in the woman uterus241. The second method used
is intracytoplasmic sperm injection (ICSI), similar to the standard IVF procedure, with the only
difference that a single sperm is injected directly into a female egg241,244. However, these
helpful and advanced procedures are not free from potential risks, such as miscarriage and
genetic problems in the foetus245,246. The genetic risks are more probable in ICSI than IVF,
because sperm cells are selected only by morphological assessment247. The UK IVF techniques
success rate in 2010 was 32.2% for women aged under 35 and 27.7% for women aged between
35-37 (source NHS). Since the introduction of ICSI methods, no important advancements have
been made in the development of diagnostic tools able to select good sperm cells, not carrying
genetic dysfunctions. Therefore, the development of a platform able to dynamically reveal or
hide progesterone molecules upon the application of an electrical potential, to select
responsive sperm cells, would be of paramount importance in both improving the success rates
and reducing the risks of IVF techniques.
1.5 Concluding remarks
Several successful studies have been reported in the literature, about the use of
switchable surfaces able to respond to the application of a wide range of external stimuli (i.e.
thermal, chemical/biological, electrical) and already applied in numerous biological and
medical applications. Such surfaces enable the control of specific biomolecular interactions and
the modulation of cellular response193, giving a crucial contribution to biotechnology in the
understanding of relevant complex biological processes. To create effective dynamic surfaces,
it is of paramount importance to perform a careful study of the desired characteristics in the
CHAPTER 1 33
smart architectures, such as end groups, molecular spacing, dynamic backbones, and to
understand if they are affected under complex biological conditions. This will allow the creation
of devices that can closely reproduce natural processes and fully realise their potential.
To date, important progress has already been made in this stimulating research field.
Novel platforms with biomimetic features have been created that can offer valuable
contributions in the enhancement of clinical methods1,173,174.
CHAPTER 1 34
1.6 PhD aim
The aim of this PhD is to design and fabricate novel switchable surfaces, able to
selectively control specific binding of biomolecules on the surface and from then recreate the
successful switching surface on a micropattern, to achieve the control of calcium signalling in
human sperm cells.
The steps needed to achieve this aim are the following:
1) Fabricating mixed SAMs to control biomolecular interactions between biotin and
Neutravidin studying the switching efficiency of a biotin-4KC:TEGT at different ratios in
Phosphate Saline Buffer medium (PBS), upon the application of an electrical stimulus.
Then, investigate the role of the switching unit length on the molecular motion on the
surface, by analysing the switching efficiency of a shorter (biotin-2KC) and a longer
(biotin-6KC) oligopeptide. The switching systems will be also analysed by Molecular
Dynamics (MD) to have a deeper insight into the molecular dynamics. This
investigation will provide a better understanding of the relationship between the
surfactant backbone and switching efficiency (Figure 1.15).
CHAPTER 1 35
Figure 1.15 - Molecular structures and related cartoons of the oligopeptide Biotin-4KC and the
triethylene glycol-terminated thiol (TEGT) used in the mixed SAMs.
2) Fabricating mixed SAMs to control biomolecular interactions between progesterone
and its murine monoclonal antibody (mAb) by studying the switching efficiency of a
Progesterone-C7-4KC:EG6OH mixed SAMs in both Phosphate Saline Buffer medium
(PBS) and Earle’s Balanced Salt Solution (EBSS), upon the application of an electrical
stimulus. This investigation provides a better understanding of the feasibility of the
exposure control of Progesterone on the surface, needed to then control the
activation of the calcium signalling in human sperm cells (Figure 1.16).
CHAPTER 1 36
Figure 1.16 – Cartoon representation of the switchable system (top), molecular structures and
related cartoons of the oligopeptide (progesterone-C7-4KC) and the hexaethylene glycol-
terminated thiol (EG6OH) used in the mixed SAMs (bottom).
3) Perform preliminary studies on the feasibility of an orthogonal functionalisation of
gold and glass surfaces to create a switchable system, composed by Progesterone-C7-
4KC:EG6OH mixed SAMs on gold and silane-poly-D-lysine (silane-PDL) layers. Study
sperm cells attachment on silane-PDL layers to set the starting point for the
development of an innovative switchable system able to monitor sperm cell response
to progesterone in real time.
CHAPTER 2 37
Chapter 2: Surface Characterisation Techniques
Abstract: This chapter briefly describes the surface characterisation techniques commonly used
in the analysis of surfaces. To design highly-ordered surfaces, it is of predominant importance
to understand the chemical and physical properties of a SAM through a detailed and precise
characterisation. The overall information about a specific surface is the sum of several data
collected using different techniques, each of which is important to achieve a complete and
accurate understanding of the different aspects of the surface analysed. Such techniques
include contact angle measurements, ellipsometry, fluorescence microscopy, X-ray
Photoelectron Spectroscopy (XPS), Surface Plasmon Resonance (SPR) and electrochemical
techniques.
2.1 Contact Angle
Contact angle is a surface characterisation technique, used to evaluate the
hydrophilicity/hydrophobicity of a surface by using a droplet of a liquid (usually water)
deposited onto the investigated surface. A contact angle goniometer consists of a light source
to illuminate the surface, a stage to hold the surface, a syringe filled with a liquid and a camera
connected to a computer for measuring contact angle values (Figure 2.1).
CHAPTER 2 38
Figure 2.1 – Schematic representation of a contact angle goniometer
The contact angle is calculated using Young’s equation (Equation 2.1)
SV = SL + LVcosθ
Equation 2.1
Where is the surface tension (or surface free energy) and SV, SL and LV are the surface
tensions for solid-vapour, solid-liquid and liquid-vapour interfaces, respectively248. When a
droplet of liquid is deposited onto a surface, the three surface tensions are in equilibrium, as
showed by Equation 2.1 and expressed by the calculated contact angle. When a surface
possesses a hydrophilic character, the surface energy is high, and the droplet spreads onto the
surface to minimise this energy. This results in a low contact angle (<30˚). On the contrary,
when a surface possesses a hydrophobic character, the surface energy is lower, and the droplet
does not spread onto the surface, resulting in a higher contact angle (>90˚) (Figure 2.2).
CHAPTER 2 39
Figure 2.2 – Representation of contact angles (θ) formed by sessile liquid drops on a solid
surface. Representation of a) a hydrophilic surface and b) a hydrophobic surface.
The contact angle can be measured using two different methods: static and dynamic
contact angle. In the static method, a droplet of liquid is deposited onto the surface, the contact
angle is measured while the droplet volume remains constant. In the dynamic method, many
droplets of liquid are deposited onto the surface dropwise, forming one larger drop on top of
it, which is then withdrew using a needle. The advancing contact angle (θa) is measured during
the addition of the liquid, and the receding contact angle (θr) is measured during its withdrawal
(Figure 2.3).
CHAPTER 2 40
Figure 2.3 – Schematic representation of a) advancing (θa) and b) receding contact angle (θr)
used in the dynamic contact angle measurements.
The difference between the advancing and receding angle (θa-θr) is called contact angle
hysteresis (θh) and gives indications about the homogeneity of a surface. A small hysteresis
(<5˚) indicates that the surface is homogenous and well-ordered, whereas a large hysteresis
suggests the surface is contaminated, non-homogenous and/or relatively rough249.
2.2 X-ray Photoelectron Spectroscopy
XPS is a surface-sensitive quantitative technique, exploited to analyse the elemental
composition (for the top 0-10 nm of a surface), the empirical formula, and the chemical and
electronic states of elements composing a material250. The first high-energy-resolution XPS
spectrum was recorded in 1957 by Kai Siegbahn and co-workers at University of Uppsala251.
This technique uses an electromagnetic source to eject electrons from the analysed
sample252 in an ultra-high vacuum (UHV) environment.
CHAPTER 2 41
An XPS apparatus comprises an ultra-high vacuum chamber where the sample is placed,
an X-ray source, an electron collection lens, an electron energy analyser, an electron detector
and a computer where the XPS peaks are visualised (Figure 2.4)
Figure 2.4 - Cartoon representation of a XPS apparatus
When an X-ray photon (h) interacts with an electron in the K shell, a 1s photoelectron
is emitted from the surface, as a result of atoms being ionised. Subsequently, an electron from
a higher energy level (L) fills the created vacancy in the inner-shell, leading either to X-ray
fluorescence photoemission process (XPS) or radiationless process of Auger electron emission,
consisting in the de-excitation and emission of a higher-shell electron (Figure 2.5)250,252,253.
CHAPTER 2 42
Figure 2.5 – Schematic diagram of XPS process for a photoelectron emitted from the core
energy level. The subsequent relaxation process of an electron from a higher energy level
(dashed arrow) fills the created vacancy (white circle), resulting in the emission of an Auger
KLL electron (dark green circle).
When electrons are transferred, for example from the L shell to the K shell, as in the
example above, this results in a decrease of the atom potential energy253. Electrons are ejected
with discrete kinetic energies (EK)254 that can be analysed and used to derive the equation to
calculate the energy conservation (Eh) of the process (Equation 2.2),
EhEK + E + EB(i)
Equation 2.2
CHAPTER 2 43
where Eh is the X-ray energy, EK is the photoelectron kinetic energy, E is a small correction for
solid effects (work function dependent on the spectrometer and the material) and it is a
constant, and EB(i) is the electron binding energy for the ith level.
The conventional photon sources are soft X-rays (MgK X-rays, h=1253.6 eV and AlK X-rays,
h=1486.6 eV) and the detection limit of the technique is 1000 ppm (0.1 atom%)255,256.
XPS leaves the atomic nuclei being examined unchanged, however some samples can
undergo decomposition upon the exposure to an X-ray source, not allowing further analysis of
the sample. The power of the X-ray photoelectron method lies in the fact that the measured
quantity, the electron binding energy of an atom, is a function of the chemical environment of
the atom254. Each element on the analysed surface can be therefore easily identified by
analysing the XPS peaks produced by each atom.
The elements on the surface can be identified by analysing the binding energy of the
core photoelectrons. The intensity (integrated area under the photoelectron peak) is
proportional to the atom quantity in the detected volume. The exact position of a
photoelectron peak indicates the chemical state of the atom, since the binding energies of the
atom core levels are affected by its chemical environment252.
2.3 Ellipsometry
Ellipsometry is a sensitive, non-destructive, optical technique developed by Drude in
1887257,258 , to calculate the dielectric function of metals and dielectrics259. This technique
exploits plane-polarised light interacting with the surface at a certain angle (usually 70˚), to
study surfaces and thin films via thickness and morphology measurements at interfaces, up to
1000Å. The measurements are commonly carried out in the UV/VIS region, but they have also
CHAPTER 2 44
been made in the infrared region260. The surface roughness needs to be small (<50Å)261 and the
measurement has to be performed at oblique incidence at an angle that maximises the
sensitivity262. A high surface roughness would cause light scattering, which dramatically
reduces light intensity, and the ellipsometry measurements would therefore become
difficult260,263. If the light incidence was normal, it would be impossible to distinguish the p- and
s- components of the light, making the measurement impossible263. Figure 2.6 shows a
schematic representation of an ellipsometer.
Figure 2.6 – Schematic representation of an ellipsometer.
The light beam emitted by the source passes through a polariser, then the linearly
polarised beam hits the surface at a selected angle and each component is reflected with a
different phase and amplitude, as an elliptically polarised light. The reflected light passes
through a compensator that can modify the phase of the beam, to finally reach the analyser
that calculates the ratio between the reflection coefficients of the two components of the light
(Equation 2.3)264
CHAPTER 2 45
𝜌 =𝑟𝑝
𝑟𝑠= tan(𝜓)𝑒𝑖∆
Equation 2.3
where tan(ψ) is the amplitude ratio upon reflection and Δ is the phase shift (difference). They
can both be calculated using Fresnel’s equations260,263. Since ellipsometry is an indirect method,
the values of ψ and Δ cannot be used directly to calculate the optical constants of the sample,
but a layer model and an iterative procedure have to be used. The iterative procedure is called
“least-squares minimization”265 and it is used to vary the optical constants and/or thickness
parameters. The optical constants are obtained from the values of ψ and Δ that best fit the
experimental data and the parameters of the sample. In order to calculate the thickness of a
SAM, a three-phase ambient/SAM/substrate model is used, in which the SAM is assumed to be
homogeneous with a refractive index ranging between 1.45 and 1.55266. This model is based
on the Cauchy equation, which considers a SAM as a transparent layer. The thickness of the
SAM is then calculated using multi-guess iterations that provide a thickness result with the
lowest χ2 (chi-square distribution) between the measured and the calculated values of ψ and
Δ.
2.4 Fluorescence Microscopy
Fluorescence Microscopy is an optical technique, fully developed at the beginning of
the 20th century, which, instead of, or in addition to, reflection and adsorption, exploits
fluorescence and phosphorescence to analyse biological, organic and inorganic samples267,268.
This technique uses the sample itself as the light source, inducing it to fluoresce269. A
fluorescence microscope employs the capability of certain materials to emit energy as visible
CHAPTER 2 46
light if irradiated with a light beam of a specific wavelength. The studied specimen can be
naturally fluorescent (e.g. chlorophyll and some minerals) or it can be labelled with a
fluorescing molecule (i.e. fluorescent dye or fluorochrome)267–270. This instrument uses a light
source at a much higher intensity than a conventional microscope. The fluorescent species
present in the sample are excited and emit light at lower energy but longer wavelength that
produces the magnified image, instead of the light source. A fluorescence microscope is
equipped with special filters designed to isolate and manipulate two distinct sets of excitation
and fluorescence emission wavelengths (Figure 2.7)269.
Figure 2.7 – Schematic illustration of a fluorescence microscope setup.
The excitation filter selects only the shorter radiation wavelengths coming from the
light source which are able to excite the fluorescing material, and send these wavelengths to
the sample, while the band of longer wavelengths emitted by the sample itself form an image
CHAPTER 2 47
of the sample, recorded by the objective269. In order to make the microscope work effectively,
fluorophores, filters and light source have to be carefully selected for a given application and
the quality of the fluorescence signals have to be analysed271.
Fluorescence microscopy is widely used to study the characteristics, functions and
intracellular distribution of numerous metabolites and biomolecules. These applications
include the use of fluorescently labelled metabolites, ligands and proteins for the interaction
with cell membranes, which in turn become fluorescently labelled271. Fluorescent antibodies
and proteins (e.g. green fluorescent protein, GFP) can also be used to tag target molecules, and
special dyes have also been developed to label organelles and cytoskeletal proteins
selectively272–276. By using radiometric dyes, it is possible to monitor the concentration of
numerous intracellular ionic species, such as Na+, K+ and Ca2+.277. Advancements in fluorescence
microscopes have led to the development of novel fluorescence microscopy techniques, such
as FRET, FRAP and TIRF. FRET, or Fluorescence Resonance Energy Transfer, is used to generate
fluorescence signals sensitive to molecular conformation, association, and separation in the 1–
10 nm range278. In this case, two different fluorophores are employed, and when the
fluorophore with the shorter wavelength is excited, this causes the excitation of the longer-
wavelength fluorophore if the two labelled moieties are separated only by a short molecular
distance (in the range 1-10 nm). FRAP, or Fluorescence Recovery After Photobleaching,
fluorescence is measured as a function of time and space, and the information about the
diffusion coefficients and the binding constants of macromolecules can be recorded279. TIRF,
or Total Internal Reflection Fluorescence microscopy, is an extremely sensitive technique,
employed in the study of molecular events occurring in the close vicinity of the membrane in
living cells, due to the short penetration distance (around 100 nm) of this type of microscopy280–
282.
CHAPTER 2 48
2.5 Surface Plasmon Resonance (SPR)
Surface plasmons were observed for the first time in 1902 by Wood283–285, who
described the phenomenon of anomalous diffraction after illuminating a metallic diffraction
grating with polychromatic light286. Further work by Fano287 showed that the narrow dark
bands in the diffracted light reported by Wood were associated with the excitation of
electromagnetic waves on the surface of the grating286,288,289.
In 1968 Otto, Kretschmann and Raether demonstrated that the drop in reflectivity in
the Attenuated Total Reflection method (ATR) observed by Thurbadar ten years earlier288–290
was due to the excitation of surface plasmons. When a p-polarised monochromatic light beam
interacts with a metal surface evaporated onto a glass prism at a certain angle (surface plasmon
resonance angle, θSP), a surface plasmon wave (SPW) is generated at the interface between the
glass prism, characterised by a high refractive index (RI), and the external medium (gas or
liquid), characterised by a low RI286,288–290.
In the Kretschmann geometry of the ATR method, a prism with a high refractive index
(np) is interfaced with a thin metal film with dielectric constant m, thickness q and a semi-
infinitive dielectric with a low refractive index nd (nd<np). When a light wave propagating in the
prism hits the metal film, part of the light is reflected back into the prism and part propagates
in the metal (Surface Plasmon Wave, SPW) (Figure 2.8).
CHAPTER 2 49
Figure 2.8 - Kretschmann geometry of the Attenuated Total Reflection (ATR) method. θSP is the
angle at which the incident light is able to excite the surface plasmon wave (SPW) at the
metal-dielectric interface, d is the thickness of the metal surface (usually 50 nm) and εm and εd
are the dielectric constants of the metal and the dielectric, respectively.
An SPW is characterised by the propagation constant () and the electromagnetic field
distribution (Equation 2.4)
𝛽 =𝜔
𝑐√
𝜀𝑚𝜀𝑑𝜀𝑚 + 𝜀𝑑
Equation 2.4
whereis the angular frequency, c is the speed of light in vacuum, m is the dielectric constant
of the metal (m=mr+imi) and d is the dielectric constant of the dielectric290–292. The excitation
of surface plasmons can be possible only if the dielectric constant of the metal (m) has a large
negative real part at the light wavelength used. This phenomenon can therefore occur only if
CHAPTER 2 50
at the interface between the two media a so-called “free electron-like” metal is present
(generally gold) and its thickness should be a fraction of the wavelength of the incident light
(usually 50 nm). Outside the metal, an evanescent electric field that decays exponentially with
distance from the metal surface is present, in the direction perpendicular to the prism-metal
surface, and interacts with the close vicinity of the metal287. The evanescent formed wave
penetrates through the metal film and couples with a surface plasmon at the outer boundary
of this film. The electromagnetic field of a surface plasmon wave is distributed in an asymmetric
manner, and the majority of this field is concentrated in the dielectric. If changes of the optical
properties of this region occur, i.e. when an analyte molecule in solution binds to a molecule
absorbed on the metal surface, the refractive index at the surface will increase, the SPR angle
will be affected and its variations will result in a change of the SPR signal recorded on the
computer screen (Figure 2.9)287,293.
CHAPTER 2 51
Figure 2.9 – Illustration of the SPR sensing principle, using the Kretschmann optical
configuration. a) The SPR detects the angle of reflection of the light when no analyte is bound
to the SAM on the surface; b) when the analyte is present in solution, this comes into contact
with the target molecules on the surface. The binding causes a variation in the mass present
on the surface, therefore a shift in the angle of reflection (c) occurs, and a change in the
intensity of the reflected is recorded by the photodiode array detector. d) A sensorgram is
obtained by plotting the resonance angle signal against time, and it is used to monitor the
changes occurring on the surface.
Surface Plasmon Resonance is nowadays fully exploited to follow the interaction
between molecules immobilised on the metal surface (molecular recognition elements) and
analyte molecules in solution (biorecognition element)294.
CHAPTER 2 52
The analyte molecules interact with their relative biorecognition element on the sensor
surface, this causes an increase in the refractive index and this change can be measured in real-
time. When the buffer solution flows again on the surface, this causes a drop in the refractive
index and the occurred variations correspond to a change in the SPR signal recorded. If the
nature of the interaction is known, the amount of analyte molecules bound to the
biorecognition elements can be calculated from the change of the SPR response units (RU).
SPR was used for the first time for biosensing in 1983 by Liedberg and his collaborators,
to investigate the interaction between an immunoglobulin (IgG) absorbed on a silver surface
and its relative antibody (anti-IgG). This interaction was demonstrated by recording a shift in
the surface plasmon angle and detecting a change in photocurrent when the antibody was
injected over the surface283,295,296.
SPR instrumentation is easy to use, and the sensing chip can usually be regenerated, to
which biomolecules could be coupled using known coupling chemistry. The possible
applications of SPR for biosensing have been extensively developed since the first
demonstration of immunosensing. The application of SPR for biosensing has been extensively
developed since the first work of Liedberg et al. SPR biosensors are a label-free real-time
analytical technology, and its major application areas include the detection of biological
analytes and the analysis of biomolecular interactions294,297.
In this research work, surface plasmon resonance, coupled to electrochemistry, was
exploited to investigate, in real-time, the interaction between neutravidin molecules in solution
and biotin moieties on the sensor surface, and between anti-mouse progesterone antibodies
in solution and progesterone moieties on the surface.
CHAPTER 2 53
2.6 Electrochemical techniques
Electrochemical techniques are used to study processes occurring when an electric
potential is applied. The most commonly used electrochemical methods are
chronoamperometry, linear sweep voltammetry and cyclic voltammetry. They will be briefly
described in the following sections.
2.6.1. Chronoamperometry
Chronoamperometry is a sensitive electrochemical technique in which the working
electrode potential (V) is changed in one step from V1 (equilibrium state) to V2, and then kept
to this potential for a defined amount of time. The resulting steady state current, caused by
the potential step, is measured as a function of time298–301. The potential stepping and the
resulting current are shown in Figure 2.10.
Figure 2.10 – Illustration of a) chronoamperometry potential stepping and b) current variation
with time
Changes in the current come from the variations in the diffusion layer at the electrode.
The concept of “diffusion layer” was introduced by Nernst, and describes the presence of a thin
layer of solution in contact with the electrode surface298,302. The local analyte concentration at
CHAPTER 2 54
the electrode surface falls to zero, and the movement of the analyte from the bulk solution of
higher concentration is controlled by diffusion. This creates a concentration gradient away
from the electrode surface, whereas the convective transfer maintains the concentration of
the analyte in the bulk solution constant. Chronoamperometry is often coupled to other
techniques such as cyclic voltammetry, for time-dependent system characterisation302.
2.6.2. Linear Sweep Voltammetry
Linear sweep voltammetry (LSV) is a voltammetric method in which the current at a
working electrode is recorded, while the potential between the working and the reference
electrode is linearly varied with time (Figure 2.11)303,304.
Figure 2.11 – Series of linear sweep voltammograms recorded at different scan rate
V1 is the lower limit of the voltage range applied, at which no reaction occurs, whereas
V2 represents the upper limit. By calculating the slope of the line, it is possible to obtain the
CHAPTER 2 55
voltage scan rate (v = Δy/Δx). When the potential starts to be swept towards V2, the electrolyte
(A) present in the electrochemical cell starts to be reduced or oxidised to form the
electrochemical product (Equation 2.5)303.
A + e- A- (Reduction)
A A+ + e- (Oxidation)
Equation 2.5
The process can be understood by looking at the Nernst equation, which shows the
relationship between the concentration of species and the potential difference (Equation
2.4)303:
𝐸 = 𝐸0 −𝑅𝑇
𝑛𝐹− ln
[𝑜𝑥]
[𝑟𝑒𝑑]
Equation 2.4
where E is the applied potential difference, E0 represents the standard electrode potential, R is
the universal gas constant (R=8.314JK-1mol-1), T is the absolute temperature (in Kelvin), F is the
Faraday constant (F=9.64853x104Cmol-1) and n is the number of electrons being transferred in
the half-reactions. The voltage is varied from V1 to V2, then the equilibrium at the electrode
surface is altered and a current can be recorded303,304. At the equilibrium (V1 applied), there is
no electron transfer in the electrochemical cell. The current increases further as the potential
is swept towards V2, due to a greater number of electrons being transferred in the system. The
result is a shift of the equilibrium towards the product (conversion of more electrolyte A),
reaching the full conversion of the analyte at the electrode when the potential applied is equal
CHAPTER 2 56
to V2. At the redox peak potential (Vp), the current reaches its maximum value, due to the
diffusion layer having sufficiently grown above the surface of the electrode. This phenomenon
makes the movement of reactant to the electrode too slow to satisfy the Nernst equation,
causing a fall in the current303,305 (Figure 2.12).
Figure 2.12 – Changes in current response for voltammograms recorded at different scan
rates.
If the scan rate is varied, this causes a linear change in the current response: if the scan
rate is increased, the total current increases. This event can be understood by analysing again
the diffusion layer present at the electrode surface, which will change with the voltage scan
rate. When the scan rate is slow, the voltammogram will take longer to record and the growth
of the diffusion layer will be bigger than in the case of a faster scan rate. This phenomenon will
reduce the flux of the analyte (reactant) to the electrode, leading to a smaller current compared
to higher scan rates, being this proportional to the flux303,305.
CHAPTER 2 57
2.6.3. Cyclic Voltammetry
Cyclic Voltammetry (CV) is a potentiodynamic electrochemical measurement, similar to
linear sweep voltammetry (LSV)302,304,306. In this case, the potential of the working electrode is
linearly swept versus time between two values at a fixed rate. Contrary to linear sweep
voltammetry, when the potential reaches the final value V2 the scan is reversed, and the
voltage is swept back towards the equilibrium position V1. This results in a triangular potential
cycle (Figure 2.13).
Figure 2.13 – Schematic representation of the forward and back scans in cyclic voltammetry.
The forward scan gives an identical response to that given by a LSV scan, but when the
scan is reversed, the system moves back towards the equilibrium position, and the product of
electrolysis is converted back to reactant. The current is flowing from the solution species back
to the electrode, occurring in the opposite direction to the forward scan. The voltage is
measured between the reference electrode and the working electrode, whereas the current is
CHAPTER 2 58
measured between the working electrode and the counter electrode. The current is then
plotted versus the voltage, in a graph called cyclic voltammogram (Figure 2.14)303.
Figure 2.14 – Schematic representation of a cyclic voltammogram for a reversible single
electrode transfer reaction, in the case of a solution containing only a single electrochemical
reactant. Epc and ipc are the peak potential and peak current relative to the cathode,
respectively, whereas Epa and ipa are the peak potential and peak current relative to the
anode, respectively
When the voltage is swept towards the reduction potential of the analyte, an increase
in the current occurs. The analyte starts to be reduced at the electrode surface, to form the
electrochemical product. Once the potential has passed the reduction potential value, the
current decreases, due to the reduction in the concentration of the analyte near the electrode
CHAPTER 2 59
surface. When the voltage is reversed towards V1, the formed product starts being reoxidised,
to form again the electrochemical reactant, and a current of opposite polarity is produced. This
current will first increase, to then decrease after a voltage peak has been formed, as the voltage
scan continues toward V1302,303.
If a reaction is reversible, the recorded CV presents specific characteristics:
1. The difference between the two peak potentials is (Equation 2.5)
𝛥𝐸 = 𝐸𝑝𝑎 − 𝐸𝑝𝑐 =59
𝑛𝑚𝑉
Equation 2.5
where a is relative to the anodic peak, c is relative to the cathodic peak and n is the number
of electrons being transferred in the electrochemical process, mV is millivolts.
2. The positions of peak voltage do not vary with the scan rate
3. The peak current ratio is always equal to 1, at each scan rate (Equation 2.6):
𝑖𝑝𝑎𝑖𝑝𝑐
= 1
Equation 2.6
4. The peak currents are proportional to the square root of the scan rate
The scan rate is a critical factor, because each scan has to be high enough to allow the chemical
reaction of interest to occur. The diffusion layer thickness can explain the role of the scan rate
as with the linear sweep voltammetry303.
In the case of an irreversible reaction, the electron exchange between the working
electrode and the analyte is very slow. This phenomenon causes the peak current for the
irreversible reaction to be lower than the reversible one. In this case, the peak current ratio
CHAPTER 2 60
differs from 1, and the difference between the peak potentials relative to the anode and the
cathode is greater than 59/n mV (Equation 2.7)303.
𝛥𝐸 = 𝐸𝑝𝑎 − 𝐸𝑝𝑐 >59
𝑛𝑚𝑉
Equation 2.7
This behaviour can be attributed to secondary chemical reactions occurring at the
electrode, triggered by the electron transfer306.
CHAPTER 3 61
Chapter 3 - Experimental Procedures and Protocols
Abstract: Materials, methods and experimental techniques used in this work are discussed in
this chapter, together with experimental procedures and protocols and data analysis by various
types of equipment.
3.1 Materials and Methods
3.1.1 Gold substrates
Polycrystalline gold substrates were purchased from George Albert PVD, Germany and
consisted either of a 50 nm gold layer deposited onto glass covered with a thin layer (5 nm) of
chromium as the adhesion layer (for contact angle and XPS analysis) or 100 nm gold layer on
100-4inch-silicon wafer, precoated with titanium as the adhesion layer (for ellipsometry
analysis). Polycrystalline gold substrates employed in SPR were purchased from Reichert
Technologies, USA, consisted of 49 nm gold with 1 nm chromium.
3.1.2 Glass substrates
Glass substrates approximately 1 cm by 1cm were cut from glass microscope plain slides
using a glass cutter. Glass microscope plain slides (26 mm by 76 mm, 0.8-1 mm thick) were
purchased from Thermo Fisher Scientific Ltd.
3.1.2 Silicon substrates
Silicon substrates approximately 1 cm by 1cm were cut from silicon wafers using a glass
cutter. The silicon wafers were purchased from IDB Technologies Ltd (Whitley, UK), with the
following specifications: type: N<100>; size: 76 mm; resistivity: 1-10 ohm-metre (Ω⋅m);
thickness: 381 m; polish: Single Side Polish (SSP).
CHAPTER 3 62
3.2 Chemicals
Commercially available chemicals and solvents were purchased from Aldrich Chemicals
and Fisher chemicals and used as received. The oligopeptides Biotin-2KC, Biotin-4KC, Biotin-
6KC and Progesterone-C7-4KC were synthesised by Peptide Protein Research Ltd. (Wickham,
UK) to > 95% purity and verified by HPLC and mass spectrometry. Neutravidin, Calcium
GreenTM-1, AM and Pluronic Acid F-127 (20% solution in DMSO) were obtained from Invitrogen
Life Technologies Ltd. (Paisley, UK). Purified Progesterone-3 Anti-Mouse monoclonal antibody
(Affinity Constant: 75x1010 L/M) was obtained from antibodies-online GmbH (Aachen,
Germany) and diluted with Phosphate buffered saline (PBS) solution. Phosphate buffered saline
(PBS) solution was prepared from a 10× concentrate PBS solution (1.37 M sodium chloride,
0.027 M potassium chloride and 0.119 M phosphate buffer) from Fisher BioReagents. Modified
Earle’s Balanced Salt Solution (sEBSS) (CaCl2∙2H2O 1.80 mM, KCl 5.37 mM, MgSO4∙7H2O 0.81
mM, NaHCO3 26.19 mM, NaH2PO4∙2H2O 1.01 mM, NaCl 116.36 mM, D-Glucose 5.55 mM,
Sodium Pyruvate C3H3O3Na 2.73 mM, Sodium Lactate C3H5O3Na 41.75 mM) was purchased
from Biological Industries Ltd. (Beit-Haemek, Israel). Triethylene glycol thiol (TEGT) was
synthesised by Dr. Parvez Iqbal, School of Chemical Engineering, University of Birmingham,
following a multistep route (Figure 3.1). The commercially available triethylene glycol (1) was
alkylated with alkyl bromide at reflux in basic conditions to obtain 2, that was then converted
to 3 in the presence of thioacetic acid Azobisisobutyronitrile (AIBN) heated at reflux for 1 h.
Deprotection of 3 was performed in mild acidic conditions at reflux for 4 h to obtain TEGT (4).
CHAPTER 3 63
Figure 3.1 – Multistep route for the synthesis of triethylene glycol thiol (TEGT)
The 11-(Mercaptoundecyl)hexa(ethylene glycol) (EG6OH) was purchased from Sigma
Aldrich and used as received.
The carboxyethylsilanetriol di-sodium salt, 25% in water was purchased from
Fluorochem Ltd (Hadfield, UK). and used as received. Poly-D-Lysine (PDL) was purchased from
Scientific Laboratories Supplies Ltd. (Hessle, UK) and diluted with UHQ water to a concentration
of 2 mg/ml and stored in the fridge.
3.3 Experimental Procedures
3.3.1 Surface Preparation
3.3.1.1 Cleaning of gold and glass surfaces
Both gold and glass substrates were cleaned by immersion in piranha solution (70%
H2SO4, 30% H2O2) at room temperature for 8 minutes and then rinsed with Ultra High Quality
(UHQ) water and then HPLC grade ethanol thoroughly for 1 min. (Caution: Piranha solution
reacts violently with all organic compounds and should be handled with care).
CHAPTER 3 64
3.3.1.2 Preparation of Biotin-2KC:TEGT, Biotin-4KC:TEGT and Biotin-6KC:TEGT mixed
Self-Assembled Monolayers (SAMs) on gold substrates
Clean gold substrates were immersed for 24 h in HPLC ethanol 0.1 mM solution of
oligopeptide and 0.1 mM TEGT solution containing 3% (v/v) triethylamine N(CH2CH3)3 to
prevent the formation of hydrogen bonds between the amino groups (NH2) of the oligopeptide
bound to the gold surface and the free oligopeptide in the bulk solution307. The different
oligopeptides solutions were mixed at the following volume ratios:
• Biotin-2KC:TEGT 1:40 and 1:100
• Biotin-4KC:TEGT 1:0, 1:1, 1:10, 1:40, 1:100 and 1:500
• Biotin-6KC:TEGT 1:40 and 1:2000
The substrates were rinsed with a solution of HPLC ethanol containing 10% (v/v)
CH3COOH to remove triethylamine and then with HPLC ethanol and dried under a stream of Ar.
3.3.1.3 Preparation of silane-PDL on glass substrates and silicon wafer
Either clean glass substrates, microscope plain glass slides or 1 cm2 chips cut from silicon
wafers were coated on one side with a carboxyethylsilanetriol di-sodium salt, 25% in water
layer by chemical vapour deposition for 2 h in a vacuum chamber. The coated glass surfaces
were cured for 30 minutes at 100˚C under vacuum and then left cooling to room temperature.
The substrates (1) were then immersed in a 1mM HCl solution in UHQ water for 5 minutes
under gentle shaking, to form carboxylic acid groups on the surface to obtain 2. 2 was rinsed
with UHQ water and then immersed in a solution 1:1 of
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide (EDC/NHS) for 15
minutes, under gentle shaking, to activate the carboxylic groups on the surface, PDL 2mg/ml
CHAPTER 3 65
was added to obtain a concentration of 0.5 mg/ml in the solution and left to react overnight,
under gentle shaking, to form amide bonds on the surface (3) (Figure 3.2).
Figure 3.2 – Multistep route for the functionalisation of glass or silicon substrates with silane-
PDL layers
3.3.2 Surface characterisation
3.3.2.1 Contact angle
Contact angles were determined using a home-built contact angle apparatus, equipped
with a charged coupled device (CCD) KP-M1E/K camera (Hitachi) that was attached to a
personal computer for video capture. The dynamic contact angles were recorded as micro-
syringe was used to quasi-statistically add liquid to or remove liquid from the drop. The drop
was shown as a live video image on the PC screen v1.96 (First Ten Angstroms) was used for the
CHAPTER 3 66
analysis of the contact angle of a droplet of UHQ water at the three-phase intersection. The
averages and standard errors of contact angles were determined from five different
measurements made for each type of SAM (in triplicate).
3.3.2.2 Ellipsometry
The thickness of the deposited monolayers and layers was determined by spectroscopic
ellipsometry, using either (i) a gold on silicon substrate with a gold thickness of 50 nm, or (ii)
silicon substrate. A Jobin-Yvon UVISEL ellipsometer with a xenon light source was used for the
measurements. The angle of incidence was fixed at 70˚. A wavelength range of 280-820 nm
was used. DeltaPsi software was employed to determine the layer thickness and the
calculations were based on a three-phase ambient/SAM/substrate model in which the SAM
was assumed to be isotropic and assigned a refractive index of 1.50. The thickness reported is
the average of five measurements (in triplicate), with the errors reported as standard deviation.
3.3.2.3 Surface Plasmon Resonance
SPR switching experiments were performed with a Reichert SR7000DC Dual Channel
Spectrometer (Buffalo, NY, USA) at 25˚C using a three-electrode electrochemical cell and a
Gamry PCI4/G300 potentiostat. The SAMs prepared on Reichert gold served as the working
electrode, the counter electrode was a Pt wire, and a standard calomel electrode (SCE) was
used as the reference electrode (Figure 3.3).
CHAPTER 3 67
Figure 3.3 – Cartoon representation of the SPR electrochemical cell
Prior to the binding studies between Biotin and Neutravidin, the sensors chips were
equilibrated by flowing either degassed PBS at 50 l/min, followed by application of -0.4 V,
open circuit or +0.3 V conditions for 10 min while passing degassed PBS through the
electrochemical cell at the flow rate of 50 l/min. While still applying a potential, neutravidin
(250 l, 37 g/ml) was injected over the sensor chip surface for 10 secs at 1500 l/min and
then 30 min at 8 l/min (the decrease in flow rate from 1500 to 8 l/min ensures that sufficient
exposure time was provided for binding to occur between the biotin on the surface and
Neutravidin in solution). To remove any unbound material, the sensor chips were washed with
degassed PBS for 10 secs at a flow rate of 1500 l/min, followed by 10 min at a flow rate of 50
l/min while still applying a potential to the chips. The same procedure was used for OC
experiments without applying a potential.
CHAPTER 3 68
Prior to the binding studies between Progesterone and its relative anti-mouse Antibody,
the sensors chips were equilibrated by flowing EBBS at 50 l/min, followed by application of -
0.4 V, open circuit or +0.3 V conditions for 10 min while passing either degassed PBS through
the electrochemical cell at the flow rate of 50 l/min. While still applying a potential, the
antibody (250 l, 37 g/ml) was injected over the sensor chip surface for 30 min at 8 l/min,
followed by 15 min at 50 l/min while still applying a potential, to allow any dissociation of the
antibody from the surface. The same procedure was used for OC experiments without applying
a potential.
3.3.2.4 X-ray Photoelectron Spectroscopy
Analysis of biotin-nKC:TEGT (n = 2, 4, 6) mixed SAMs: XPS spectra were obtained on the
VG Escalab 250 instrument based at University of Leeds EPSRC Nanoscience and
Nanotechnology Facility, UK. XPS experiments were carried out using a monochromatic Al K α
X-ray source (1486.7 eV) and a take-off angle of 15°. High-resolution scans of N (1s) and S (2p)
were recorded using a pass energy of 150 eV at a step size of 0.05 eV. Fitting of XPS peaks was
performed using the Avantage V 2.2 processing software. Sensitivity factors used in this study
were: N (1s), 1.73; S (2p), 2.08; Au (4f 7/2), 9.58; Au (4f 5/2), 7.54. The averages and standard
errors reported were determined from at least four different XPS measurements.
Analysis of progesterone-C7-4KC:EG6OH mixed SAMs and silane layers: XPS spectra
were obtained on the AXIS Nova (Kratos Analytical) instrument based at University of
Newcastle (NEXUS), UK. XPS experiments were carried out using a monochromatic Al Kα X-ray
source (1486.7 eV) at a take-off angle of 90 degree to the surface plane. High-resolution scans
of Au4f, C 1s, O 1s, N 1s, S (2s and 2p) and Si (2s and 2p) were recorded using pass energy of 20
CHAPTER 3 69
eV at a step size of 0.1 eV. Fitting of XPS peaks was performed using CASA XPS processing
software. Sensitivity factors used in this study were: C 1s 1.00; O 1s 2.93; N (1s), 1.80; S (2s),
2.43; S (2p), 2.08; Au (4f), 17.10; Si (2s), 0.95; Si (2p), 0.82.
3.3.2.5 Force Field Test
The Force Field Test was conducted by Dr Xingyong Wang at Nanjing University, China.
Since the conformational switching of biotin-nKC chains mainly results from the rotation
of the C-C bonds, the energy scan for biotin-4KC molecule with different C1-C2-C3-C4 dihedrals
(θ) was carried out by both force field methods and density functional theory (DFT) calculations
with the B3LYP functional and 6-31G(d) basis set. Three kinds of force fields, cvff, compass and
pcff were tested. The result is shown in Figure 3.4. The cvff force field shows the best
performance. Although it overestimates the energies compared to the DFT result, it displays
the right shape of the energy curve. In contrast, both compass and pcff force fields result in a
significant deviation from the DFT result. So, the cvff force field was adopted throughout our
simulations (Table 3.1).
CHAPTER 3 70
Figure 3.4 - The energy scanning for biotin-4KC molecule with different C1-C2-C3-C4 dihedrals,
θ, obtained by both force field methods and DFT calculations.
Table 3.1 - Parameters for the surface models used in the simulations.
Surface chains Solvent molecules
(H2O)
Ions
(Cl-) Cell parameters (Å3)
Biotin-2KC/8(TEGT) 957 2 25.95 × 25.95 × 65.42
Biotin-4KC/15(TEGT) 2115 4 34.60 × 34.60 × 77.42
Biotin-6KC/15(TEGT) 2728 6 34.60 × 34.60 × 95.42
9(Biotin-4KC) 1982 36 34.60 × 34.60 × 77.42
CHAPTER 3 71
3.2.2.6 Computational details
Five layers of gold atoms cut from the Au (111) surface were adopted to model the gold
substrates used in the experiment and they were fixed during the simulations. All MD
simulations were performed in the canonical (NVT) ensemble using the cvff force field. The
temperature was set to 298 K by using the Andersen thermostat308. The equations of the
motion were integrated using the velocity Verlet algorithm309 with the time step of 1fs. The
atomic charges for the biotin-nKC molecules were updated every 100ps by DFT calculations, at
the M06-2X/6-31G(d,p) level of theory. The Discover module in the Materials Studio package310
was employed to run all the MD simulations. All DFT calculations were carried out with the
Gaussian 09 program package311.
3.3 Preparation and Analysis of Sperm Cells
3.3.1 Sperm Cells Incubation and labelling
Sperm cells were prepared at Birmingham Women’s Hospital, using the facilities at the
Dr Kirkman-Brown’s research laboratory. Cells were isolated from seminal plasma using the
following procedure: 1 ml of modified Earle’s Balanced Salt Solution (sEBSS) containing 0.3%
of Bovine Serum Albumin (BSA) were pipetted into a series of 5 ml test tubes. Volumes of 300
l of fresh semen sample, stored in the incubator at 37°C and 6% CO2 for no more than 30 min
after production, were deposited at the bottom of each test tube. After 1-hour incubation
(37°C; 6% CO2) the top 700 l of the swim-up suspension is gently removed from each tube and
transferred to clean loose-capped test tubes for capacitation. The sperm cells are finally left in
the incubator (37°C; 6% CO2) for at least 3 hours, to allow the capacitation process312. After the
capacitation was completed, 3 ml of a solution composed by 8 l of Calcium GreenTM-1, AM
CHAPTER 3 72
and 42 l of Pluronic Acid F-127 (20% solution in DMSO) were added to 300 l of sperm cells
suspension. The sperm cells were then left in the incubator (37°C; 6% CO2) for 45 min to allow
the correct labelling of sperm cells.
3.3.2 Sperm Cells Counting
10 l of swim-up suspension were deposited in a Neubauer chamber and sperm cells
were counted using the Computer Assisted Sperm Analysis (CASA) System313.
3.3.3 Perfusion Chamber
The polycarbonate perfusion chamber (dimensions 51 mm x 30 mm x 25 mm) was made
by Mr Stephen Brookes (School of Physics & Astronomy) and presents openings for the buffer
flow in and out of the chamber and the microscope objective to analyse the cell adhesion to
the glass surfaces (Figure 3.5).
Figure 3.5 – Picture of the polycarbonate perfusion chamber. The labels correspond, from left
to right, to the openings for the buffer perfusion inside the chamber, the microscope objective
and the buffer flowing out of the chamber.
CHAPTER 3 73
3.3.4 Fluorescence Microscopy
The fluorescence image time-lapses were acquired on an Olympus BX60M upright
microscope, using an Olympus LUMPlanFL 40x/0.80 W dipping objective. Samples were
illuminated with a Cairn OptoLED LED (470 nm), filtered to 480/40 nm, and emission
fluorescence, filtered to 535/50nm, was captured with a Photometrics QuantEm:512SC
Camera.
CHAPTER 4 74
Chapter 4 - Study of the effect of switching unit length on switching ability
This chapter is based on the manuscript “Modulation of Biointeractions by Electrically
Switchable Oligopeptide Surfaces: Structural Requirements and Mechanism” by C. L. Yeung, X.
Wang, M. Lashkor, E. Cantini, F. J. Rawson, P. Iqbal, J. A. Preece, J. Ma, P. M. Mendes, Advanced
Materials Interface, 2014, 1, 1300085. (My personal contribution to this work regarded the
formation of mixed Self-Assembled Monolayers on gold substrates, their characterisation by
XPS and the study of mixed monolayers switching properties by electrochemical SPR).
Abstract: This chapter presents a detailed analysis of the development of switchable mixed self-
assembled monolayers exploiting charged oligopeptides as switching units. A model system
composed by a biotinylated lysine oligopeptide has been used for the purposes of this work.
Herein a detailed study is presented, on the influence of the switching unit length on the mixed
SAMs performance under an electrical potential. The role of triethylene-glycol (TEGT)
molecules, used as second component in the mixed SAMs, has also been investigated. The
desired results have been obtained by testing different biotinylated oligopeptides/TEGT ratios
on the gold surfaces. TEGT molecules are fundamental to both confer protein-resistant
properties to the surface, to prevent any Neutravidin unspecific binding, and ensure enough
space for the switchable units to efficiently undergo their molecular rearrangement on the gold
surface, upon the application of an electrical potential. Furthermore, molecular dynamics
simulations have been conducted on the mixed SAMs employed in this study, to have a detailed
understanding of the dynamics regulating the molecular activity on the surface. This work
creates the basis for the design of efficient switchable materials, that can be employed in the
control of complex biological processes.
CHAPTER 4 75
4.1 Introduction
The possibility of modulating the surface properties by applying different stimuli has
attracted research interest in the past decades1,173. By changing the surface characteristics
using temperature314,315, light316,317, magnetic field318 and electrical potential174,194,196,208,319, is
possible to create devices with tailored features, able to mimic biological processes1,173 and
have potential biomedical applications9,10,320.
The range of applications of stimuli-responsive surfaces is wide and includes drug
delivery systems321,322, biosensors210,323,324, separation, bioanalysis and microfluidic systems325–
328, but also regenerative medicine1,173. Among the different variety of stimuli-responsive
surfaces, self-assembled monolayers able to rearrange themselves upon the application of an
electrical potential, have gained growing interest as devices able to selectively control
biomolecular interactions174,194,196,198,199. The noticeable characteristics of these smart surfaces
are a fast response time and the easiness of orthogonal functionalisation1,173,329, allowing the
creations of different switchable regions on the same substrates. Another important feature is
the possibility of inducing conformational changes on the surface, by using biologically
compatible low voltages or electric fields330. These monolayers have been shown to be able of
regulating the interactions between proteins196,208,319, DNA331,332 and both mammalian199,330
and bacterial195 cells, and the biomolecules on the surface. Specifically, oligopeptides can be
employed as switching units. Charged aminoacid, such as lysine or aspartic acid, present either
a positive or a negative charge on their side chains at pH 7. Chains composed by charged
oligopeptide can then undergo a molecular rearrangement on the surface, upon the application
of an electrical potential and they can be used to expose or conceal target molecules on
demand.
CHAPTER 4 76
Different bioactive moieties can be bound to the switchable units, making charged
oligopeptide SAMs an excellent tool to study biological processes in different
conditions1,173,174,208. In this work, a detailed study was conducted to analyse how both the
length of the switching unit and the mixed SAM surface ratio, can control the performance of
the smart monolayers under an electrical potential. The first component of the chosen mixed
SAM for this work, is a lysine (K) oligopeptide, functionalised at one end with a biotin moiety,
able to strongly interact with the Neutravidin protein and at the other end with an aminoacid
of cysteine (C) to anchor the oligopeptide to the gold substrates via the thiol group. The second
component is represented by a triethylene glycol-terminated thiol (TEGT). The aim of TEGT
molecules is both to space out the oligopeptide to allow enough space on the surface for
molecular rearrangement and impede any undesired unspecific binding of neutravidin protein
on the gold surfaces (Figure 4.1).
Figure 4.1- Chemical structures of the oligopeptides (biotin-2KC, biotin-4KC and biotin-6KC)
and tri(ethylene glycol)-terminated thiols used to form the mixed SAMs tested in this research
project.
The essential characteristic of oligolysine peptides is the presence positive charge on
the side chains at pH 7. This feature gives the possibility of an “ON/OFF” switching of the biotin-
CHAPTER 4 77
Neutravidin interaction on the surface upon the application of an electrical potential. It has
already been demonstrated that these SAMs can control the interaction between biotin on the
surface and neutravidin in solution196, but the nature of the molecular changes has not been
clarified yet. To understand the dynamics of the studied process, molecular dynamics
simulations have also been conducted on the developed system. The biotin-neutravidin
interactions can be followed by electrochemical SPR, that presents an advantage if compared
to the standard SPR technique. In fact, in addition to the easiness of use and the quick response
time, it offers the possibility of monitoring the changes happening on the surface when an
electrical potential is applied, in real-time, as illustrated in section 2.5.
The results of this analysis will permit a better design of switchable mixed SAMs, that
can therefore be tailored, according to the biological requirements and applications of the
desired device.
CHAPTER 4 78
4.2 Objectives
1. Characterisation of different biotin-4KC/TEGT ratios on gold surfaces by XPS, to
evaluate the differences between solution ratio and surface ratio.
2. Analysis, by electrochemical SPR, of the switching properties of the different mixed SAM
ratios, to select the one with the highest switching efficiency.
3. Comparison of biotin-4KC switching efficiency with the one of a shorter (biotin-2KC) and
a longer (biotin-6KC) oligopeptide, to analyse how the switching unit length influences
the molecular rearrangement on the surface.
4. Testing the switching properties of the different oligopeptides used in this work by
molecular dynamics simulations, to have an insight into the mechanism regulating the
molecular switching.
CHAPTER 4 79
4.2 Results and discussion
4.2.1 Formation of mixed biotin-4KC:TEGT SAMs
Piranha-cleaned gold substrates were incubated into different solution ratios of mixed
biotin-4KC:TEGT SAMs. The biotin-4KC peptide is composed by a chain of four lysines
functionalised at one end with a biotin moiety, that can bind to the Neutravidin molecules in
solution and, at the other end, with a cysteine aminoacid for the anchoring to the gold
substrate. The flexible lysine backbone presents a positive charge at pH 7, making it employable
as the mixed SAM switching unit. Upon the application of a negative potential to the gold
substrate, the lysine oligopeptide is expected to be attracted towards the surface. This
attraction will drag the biotin moiety, that will then be hindered from the interaction with
neutravidin, by the TEGT molecules. When a positive potential is applied, an opposite
behaviour is expected: the lysine oligopeptide will be repelled by the charge, being fully
extended on the surface, therefore completely exposing the biotin moiety for the interaction
with neutravidin. TEGT molecules were employed to prevent non-specific binding of proteins
on the surface and to give enough conformational freedom to the biotin-4KC component to
undergo the switching (Figure 4.2).
CHAPTER 4 80
Figure 4.2 – Schematic representation of the biotin-4KC:TEGT mixed SAMs in the bio-active
state (+0.3V), where the biotin moiety is fully exposed on the gold surface, and in the bio-
inactive state, where the biotin moiety is concealed and hindered from the binding to
neutravidin.
4.2.2 XPS characterisation of mixed biotin-4KC:TEGT SAMs
One of the objectives of this research work was the selection of the mixed biotin-
4KC:TEGT SAM ratio presenting the highest switching performance. The first step was to
characterise the different SAMs ratios by XPS, to analyse if the two components were forming
the ratio they had in solution, on the surface. In addition, the XPS analysis was important to
understand if an increase of one of the two mixed SAM components in solution, was resulting
in an increase of the same component on the surface. The different mixed SAMs were prepared
from biotin-4KC:TEGT solution ratios 1:0 (pure biotin-4KC SAM), 1:1, 1:10, 1:40, 1:100 and
1:500, by immersing piranha-cleaned gold substrates in each solution ratio for 24 h, as reported
in section 3.3.1. The XPS spectra were recorded by Dr C. Yeung and analysed by myself. The
XPS spectra of the mixed monolayers revealed the presence of signals from C 1s, O 1s, N 1s and
S 2p, confirming the formation of the mixed biotin-4KC:TEGT SAMs. For simplicity, only the
CHAPTER 4 81
spectra of nitrogen and sulphur atoms are reported, since these are the elements used to
calculate the surface ratio.
The mixed SAM surface ratio can be calculated from the integrated peaks areas, using
the equation below (Equation 4.1):
𝑁𝑢𝑚𝑏𝑒𝑟𝑜𝑓𝑇𝐸𝐺𝑇 = (𝑁𝑜𝑜𝑓𝑁𝑝𝑒𝑟𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑥𝑆𝑎𝑟𝑒𝑎
𝑁𝑎𝑟𝑒𝑎) − 𝑁𝑜𝑜𝑓𝑆𝑝𝑒𝑟𝑝𝑒𝑝𝑡𝑖𝑑𝑒
Equation 4.1
As seen from the molecular structure of biotin-4KC (Figure 4.1), 11 nitrogen atoms are
present, whereas the number of sulphur atoms is only 1. TEGT molecules do not possess any
nitrogen atom, but only a sulphur atom. Using the Equation 4.1 is possible to estimate the
number of sulphur atoms remaining when the sulphur from cysteines are subtracted,
corresponding to the number of TEGT molecules. Table 4.1 shows an example of how the
surface ratio was calculated from the solution ratio, using Equation 4.1.
Table 4.1 – XPS data of biotin-4KC:TEGT 1:10 solution ratio and average number of TEGT
molecules per biotin oligopeptide on the surface. The calculated average and error are based
on three experiments (a, b and c).
Solution
ratio
N area S area S/N No. of
TEGT
Rounded
No of TEGT
Average Error
1:10 a 0.00691 0.00538 0.778 6.564 7
5 2 1:10 b 0.00653 0.00365 0.559 4.148 4
1:10 c 0.00688 0.00402 0.584 4.427 4
CHAPTER 4 82
The first tested mixed biotin-4KC:TEGT SAM ratio was 1:0, that corresponds to a SAM
by biotin-4KC molecules only. The S 2p spectrum (Figure 4.3) can be deconvoluted into two
doublet peaks86,333,334. The first doublet peak is centred at 163.3 eV (S 2p1/2) and 162.0 eV (S
2p3/2), corresponding to the sulphur chemisorbed to the gold substrate. The second doublet
peak, attributed to S-C bonds on the biotin moieties, is centred at 164.1 eV and 165.3 eV,
respectively86,333,334. The N 1s spectrum (Figure 4.3), presents a large peak centred at 400.0 eV,
corresponding to amino (NH2) and amide (CONH) groups333–335. The second, smaller peak
centred at 402.0 eV can be attributed to charged amino groups (NH3+), present on lysine side
chains333–335.
Figure 4.3 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:0 solution ratio
The second ratio of mixed biotin-4KC:TEGT SAM tested was 1:1. The S 2p spectrum
(Figure 4.4) presents two doublets peaks, the first one centred at 163.2 eV (S 2p1/2) and 162.0
eV (S 2p3/2), refers to the sulphur chemisorbed on the gold substrates, whereas the second
doublet peaks, centred at 163.8 eV and 165.0 eV, respectively, can be assigned to S-C bonds
present on biotin moieties. As expected, the N 1s spectrum (Figure 4.4) presents a smaller peak,
compared to the 1:0 ratio, due to a smaller number of biotin-4KC molecules on the surface.
395400405Binding Energy (eV)
1:0 Biotin-4KC:TEGT mixed SAMs
69
160164168Binding Energy (eV)
1:0 Biotin-4KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 83
The first peak, corresponding to amino (NH2) and amide (CONH) groups, is centred at 400.3 eV,
whereas the smaller peak centred at 402.2 eV is assignable to charged amino groups (NH3+),
present on lysine side chains333–335. By using Equation 3.1, it is possible to calculate the mixed
biotin-4KC:TEGT SAM surface ratio. The calculated surface ratio does not directly correspond
to the solution ratio, in fact it is equal to 1:3±3 (Table 4.2). This discrepancy has been reported
before for other mixed monolayers79,80,336. This trend has been observed also for the all the
solution ratios tested.
Figure 4.4 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:1 solution ratio
The third biotin-4KC:TEGT solution ratio analysed by XPS was 1:10. The S 2p spectrum
(Figure 4.5) presents two doublets peaks, the first one centred at 163.4 eV (S 2p1/2) and 162.0
eV (S 2p3/2), refers to the sulphur chemisorbed on the gold substrates, whereas the second
doublet peaks, centred at 163.7 eV and 165.2 eV, respectively, can be assigned to S-C bonds
present on biotin moieties. As expected, the N 1s spectrum (Figure 4.5) presents a smaller peak,
compared to the 1:1 ratio, due to a smaller number of biotin-4KC molecules on the surface and
the charged amino group peak start to be difficult to be identified. The first peak, centred at
400.4 eV, can be assigned to amino (NH2) and amide (CONH) groups, whereas the smaller peak
395400405Binding Energy (eV)
1:1 Biotin-4KC:TEGT mixed SAMs
67
160164168Binding Energy (eV)
1:1 Biotin-4KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 84
centred at 401.8 eV is ascribable to charged amino groups (NH3+), present on lysine side
chains333–335. Again, by using Equation 4.1, it is possible to calculate the mixed biotin-4KC:TEGT
SAM surface ratio. As expected, the calculated surface ratio does not directly correspond to
the solution ratio, in fact it is equal to 1:5±2 (Table 4.2).
Figure 4.5 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:10 solution ratio
The fourth biotin-4KC:TEGT solution ratio analysed by XPS was 1:40. The S 2p spectrum
(Figure 4.6) presents two doublets peaks, the first one centred at 163.4 eV (S 2p1/2) and 162.1
eV (S 2p3/2), refers to the sulphur chemisorbed on the gold substrates, whereas the second
doublet peaks, centred at 163.7 eV and 165.0 eV, respectively, can be assigned to S-C bonds
present on biotin moieties. As expected, the N 1s spectrum (Figure 4.6) presents a smaller peak,
compared to the 1:10 ratio, due to a smaller number of biotin-4KC molecules on the surface
and the peak corresponding to charged amino groups is barely identifiable. The first peak,
centred at 400.5 eV, can be assigned to amino (NH2) and amide (CONH) groups, whereas the
smaller peak centred at 402.0 eV is ascribable to charged amino groups (NH3+), present on
lysine side chains333–335. Again, by using Equation 4.1, it is possible to calculate the mixed biotin-
395400405Binding Energy (eV)
1:10 Biotin-4KC:TEGT mixed SAMs
73
160164168Binding Energy (eV)
1:10 Biotin-4KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 85
4KC:TEGT SAM surface ratio. As expected, the calculated surface ratio does not directly
correspond to the solution ratio, in fact it is equal to 1:16±4 (Table 4.2).
Figure 4.6 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:40 solution ratio
The fifth biotin-4KC:TEGT solution ratio analysed by XPS was 1:100. The S 2p spectrum
(Figure 4.7) presents two doublets peaks, the first one centred at 163.2 eV (S 2p1/2) and 162.0
eV (S 2p3/2), refers to the sulphur chemisorbed on the gold substrates, whereas the second
doublet peaks, centred at 163.6 eV and 164.8 eV, respectively, can be assigned to S-C bonds
present on biotin moieties. As expected, the N 1s spectrum (Figure 4.7) presents a smaller peak,
compared to the 1:10 ratio, due to a smaller number of biotin-4KC molecules on the surface
and the peak corresponding to charged amino groups cannot be identified. The peak centred
at 400.3 eV, can therefore be assigned to amino (NH2) and amide (CONH) groups only333–335.
Again, by using Equation 3.1, it is possible to calculate the mixed biotin-4KC:TEGT SAM surface
ratio. As expected, the calculated surface ratio does not directly correspond to the solution
ratio, in fact it is equal to 1:22±8 (Table 4.2).
395400405Binding Energy (eV)
1:40 Biotin-4KC:TEGT mixed SAMs
63
160164168Binding Energy (eV)
1:40 Biotin-4KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 86
Figure 4.7 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:100 solution ratio
The sixth biotin-4KC:TEGT solution ratio analysed by XPS was 1:500. The S 2p spectrum
(Figure 4.8) presents two doublets peaks, the first one centred at 163.3 eV (S 2p1/2) and 162.1
eV (S 2p3/2), refers to the sulphur chemisorbed on the gold substrates, whereas the second
doublet peaks, centred at 163.3 eV and 164.5 eV, respectively, can be assigned to S-C bonds
present on biotin moieties. The only N 1s peak recorded (Figure 3.8), centred at 400.4 eV, can
be ascribed to amino (NH2) and amide (CONH) groups333–335.
Figure 4.8 – XPS spectra of N 1s (left) and S 2p (right) for biotin-4KC:TEGT 1:500 solution ratio
68
160164168Binding Energy (eV)
1:100 Biotin-4KC:TEGT mixed SAMs
70
160164168Binding Energy (eV)
1:500 Biotin-4KC:TEGT mixed SAMs
N (1s) S (2p)
N (1s) S (2p)
CHAPTER 4 87
As expected, the calculated surface ratio does not directly correspond to the solution
ratio, in fact it is equal to 1:38±6 (Table 4.2).
Table 4.2 – Biotin-4KC:TEGT solution ratios and respective surface ratios calculated after XPS
analysis.
Biotin-4KC:TEGT ratio
Solution Surface
1:0 1:0
1:1 1:3±3
1:10 1:5±2
1:40 1:16±4
1:100 1:22±8
1:500 1:38±6
The results obtained always showed a difference between the biotin-4KC:TEGT solution
ratios and the surface ratio calculated from the XPS nitrogen and sulphur peaks. The averages
and standard errors reported in the calculated surface ratios are the result of at least four
different XPS measurements. All the samples were reproducible. By using correlation analysis,
it is possible to find a logarithm relationship between the solution and the surface ratios of
mixed biotin-4KC:TEGT SAMs, with the biotin-4KC surface composition being significantly
higher than it solution composition. This result is in agreement with previous studies on mixed
SAM composed by different chain lengths thiols, showing a preference in the absorption of the
longer chain thiol79,80,336. The exact reason for this phenomenon is still unclear337. Mixed SAMs
can present microscopic phase separation80,337, where the two components are not randomly
CHAPTER 4 88
dispersed throughout the monolayer. The longer alkyl-thiol molecules have to adopt complex
conformation and tilt at an angle greater than 30˚ to the surface normal to maximise Van der
Waals interactions80. In this way, the alkyl chains near the gold surface adopt a more disordered
conformation than in pure alkanethiolate SAMs. On the contrary, the molecules of the shorter
alkanethiolate component, are organised in a highly-oriented form80, similar to the one they
present in a pure SAM. In addition, long alkyl chain tend to organise in a gauche conformation,
in which the backbone chains are not orderly-packed over the surface80. As stated in section
1.3.3, alkyl-thiols composing mixed SAMs are motile, feature resulting in a dynamic adsorption
and desorption of the molecules of excess adsorbate80. The preferential absorption of longer
thiol molecules it is due to thermodynamically cohesive interactions between the alkyl
chains.337 The rate of this thermodynamic process increases with the molecular length of the
component and as the chain length increases, the preferential adsorption of the longer thiol
increases as a consequence337.
4.2.3 Investigation on biotin-4KC:TEGT binding capacity and switching efficiency
To understand if the biotin-4KC:TEGT mixed SAMs could control the biotin bioactivity
upon the application of an electrical stimulus, the different solution ratios, ranging from 1:1 to
1:500 and previously analysed by XPS, were tested by electrochemical SPR. The pure biotin-
4KC SAM was also tested. Switching studies were conducted by monitoring the Neutravidin
binding to the biotin end groups on the oligopeptide. Previously, our research group had
already demonstrated that oligopeptide mixed SAM can be employed to modulate
bioactivity196, upon the application of an electrical potential, while not affecting the SAM
integrity. Thus, similar potentials were used in this research work. The SPR experiments were
CHAPTER 4 89
conducted in three different states: bio-active (ON), while a positive potential of +0.3 V was
applied, open circuit (OC) while no potential was applied and bio-inactive (OFF), while a
negative potential of -0.4 V was applied. The recorded sensorgrams are reported in Figure 4.9.
Figure 4.9 – SPR sensorgram traces showing the binding of neutravidin to the biotin-4KC:TEGT
mixed SAMs at solution ratios of 1:0, 1:1,1:10, 1:40, 1:100 and 1:500 under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
Phosphate Buffer Saline (PBS) was flushed over the surface for 10 minutes to equilibrate
the sensor chip and set the SPR baseline, in either ON (+0.3 V), OC (no potential) or OFF (-0.4
V) states. This step was followed by injection of Neutravidin, diluted in PBS, over the surface
for 30 minutes, necessary for neutravidin to bind to the accessible biotin moieties on the
surface. After this exposure time, the chip was rinsed with degassed PBS, to remove any
unbound protein still present in the sensor cell and the binding capacity was recorded. The
binding capacity (BC) is defined as the difference in the SPR response units between the
beginning of Neutravidin injection and the end of washing with PBS. The averages and the
CHAPTER 4 90
standard errors in the reported binding capacity were calculated from at least three different
SPR measurements for each ratio (Table 4.3).
Table 4.3 – Biotin-4KC:TEGT Binding Capacity (BC), expressed in Resonance Units (RU) and
Switching Efficiency calculated from SPR experiments.
Biotin-4KC:TEGT ratio Binding Capacity (RU) Switching Efficiency (%)
Solution Surface
1:0 1:0 3553±258 7±2
1:1 1:3±3 3192±164 27±3
1:10 1:5±2 3053±69 34±5
1:40 1:16±4 2195±161 90±3
1:100 1:22±8 1492±72 62±8
1:500 1:38±6 1375±75 60±4
The switching efficiency (SE) was calculated as the percentage between the binding
capacity when a positive potential was applied (BCON) and the binding capacity when a negative
potential was applied (BCOFF), divided by BCON:
𝑆𝐸 =𝐵𝐶𝑂𝑁−𝐵𝐶𝑂𝐹𝐹
𝐵𝐶𝑂𝑁𝑥100
Equation 4.2
From the results obtained, it is possible to infer that a pure biotin-4KC SAM, which
presents a neutravidin immobilisation capacity of 3.5 ng/mm2 (1000 RU = 1 ng/mm2)196, is not
able to control the biotin bioactivity on the surface, showing only 7±2% of switching efficiency.
The binding capacity of the biotinylated mixed SAM decreased significantly with the reduction
CHAPTER 4 91
of biotin moieties concentration on the gold substrate. A reduced number of biotin
oligopeptide molecules on the surface, results in the decrease of neutravidin immobilisation
capacity. In fact, the 1:1 solution ratio presents a neutravidin immobilisation capacity of 3.2
ng/mm2, that is then lowered to 3 ng/mm2 and 2.2 ng/mm2 for the 1:10 and 1:40 solution
ratios, respectively. However, the diminution in the binding capacity does not show
proportionality with the reduction of the amount of biotinylated oligopeptide on the surface.
it is therefore possible to assume that both the steric hindrance and the restricted mobility of
the densely-packed oligopeptide molecules might limit the neutravidin binding to the biotin
ends at low ratio of TEGT to biotin-4KC338. The 1:16 surface ratio (1:40 solution ratio) of the
biotin-4KC:TEGT mixed SAM presented the highest switching efficiency, resulting to be the
optimum ratio for controlling the biotin bioactivity. The data collected demonstrated that a
densely-packed switchable oligopeptide SAM cannot undergo a molecular rearrangement on
the surface and some free space is needed for the oligopeptide chains to change their
conformation when a negative potential is applied (Figure 4.10).
CHAPTER 4 92
Figure 4.10 – Binding capacity and switching efficiency in ON (+0.3 V), OC (no potential
applied) and OFF (-0.4 V) states of biotin-4KC:TEGT mixed SAMs at the different solution ratios
tested in this study (1:0, 1:1, 1:10, 1:40, 1:100 and 1:500).
By progressively reducing the density of biotinylated oligopeptides on the surface, the
mixed SAM showed an increased capability of controlling the bioactivity of biotin moieties on
the surface. However, at a surface ratio lower than 1:16, the switching efficiency decreased
significantly. This phenomenon is due to the formation of a more packed ethylene glycol thiol
matrix that limit the oligopeptide free movement on the surface. Our hypothesis is that the
switching mechanism between bio-active and bio-inactive states is controlled by the
conformational changes occurring on the surface upon the application of an electrical
potential. These changes result into a reorganisation of the biotin end on the surface. In
addition, the presence of enough free space on the surface is fundamental to permit the
molecular rearrangement of bioactive molecules. From these observations, we can also
conclude that the gap distance between the bioactive molecules and the ethylene glycol thiol
CHAPTER 4 93
matrix is an important factor that has to be considered in the design of mixed switchable
monolayers. This distance should enable biotin to correctly be buried into the binding pocket
of the neutravidin barrel.
It is possible to conclude that the molecular rearrangement of oligopeptides may
induce: a) a change in the gap distance between biotin and the ethylene glycol thiol matrix, and
b) a variation in the orientation of the biotin itself that could become more or less available for
protein binding.
4.3 Study on the influence of switching unit length on switching efficiency
To investigate the influence of both the gap distance and the length of the oligopeptide
chain on the switching efficiency, a shorter (biotin-2KC) and a longer (biotin-6KC) oligopeptide
were chosen as model systems. Starting from the results obtained for biotin-4KC:TEGT mixed
SAMs at different surface ratios, two surface ratios were selected, namely 1:5 and 1:16. In
addition to evaluating the switching ability of biotin-2KC and biotin-6KC, using an
underperforming (1:5) and an optimum (1:16) ratio found for biotin-4KC aimed to a) compare
the oligopeptides behaviour and b) reveal if there is a correlation between the switchable
backbone length and the molecular area required for the molecular rearrangement to occur
under an electrical stimulus.
4.3.1 XPS characterisation of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs
The XPS spectra were recorded by Dr C. Yeung and analysed by myself. The XPS spectra
of the mixed monolayers revealed the presence of signals from C 1s, O 1s, N 1s and S 2p,
confirming the formation of both mixed biotin-2KC:TEGT and biotin-6KC:TEGT SAMs. For
simplicity, only the spectra of nitrogen and sulphur atoms are reported, since these are the
CHAPTER 4 94
elements used to calculate the surface ratio. The mixed SAM surface ratio can be calculated
from the integrated peaks areas, using Equation 4.1. The solution ratios giving the desired
surface ratios were 1:40 and 1:100, for biotin-2KC:TEGT mixed SAMs and 1:40 and 1:2000 for
biotin-6KC:TEGT mixed SAMs, respectively.
The XPS spectra for biotin-2KC:TEGT 1:40 solution ratio are reported in Figure 4.11. The
S 2p spectrum presents two doublets peaks, the first one centred at 163.3 eV (S 2p1/2) and
162.2 eV (S 2p3/2), refers to the sulphur chemisorbed on the gold substrates, whereas the
second doublet peaks, centred at 163.5 eV and 165.1 eV, respectively, can be assigned to S-C
bonds present on biotin moieties. As expected, the N 1s spectrum presents a small peak,
centred at 400 eV, can be assigned to amino (NH2) and amide (CONH) groups.
Figure 4.11 – XPS spectra of N 1s (left) and S 2p (right) for biotin-2KC:TEGT 1:40 solution ratio
The XPS spectra for biotin-2KC:TEGT 1:100 solution ratio are reported in Figure 4.12.
The S 2p spectrum presents two doublets peaks, the first one centred at 163.4 eV (S 2p1/2) and
162.0 eV (S 2p3/2), can be assigned to the sulphur chemisorbed on the gold substrates, whereas
the second doublet peaks, centred at 163.7 eV and 165.4 eV, respectively, refers to S-C bonds
74
84
94
395400405Binding Energy (eV)
1:40 Biotin-2KC:TEGT mixed SAMs
38
160164168Binding Energy (eV)
1:40 Biotin-2KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 95
present on biotin moieties. The N 1s spectrum presents a single, small peak, centred at 400.2
eV, can be assigned to amino (NH2) and amide (CONH) groups.
Figure 4.12 – XPS spectra of N 1s (left) and S 2p (right) for biotin-2KC:TEGT 1:100 solution
ratio
The XPS spectra for biotin-6KC:TEGT 1:40 solution ratio are reported in Figure 4.13. The
S 2p spectrum presents two doublets peaks, the first one centred at 163.3 eV (S 2p1/2) and
161.8 eV (S 2p3/2), is ascribable to the sulphur chemisorbed on the gold substrates, whereas
the second doublet peaks, centred at 163.6 eV and 165 eV, respectively, can be assigned to S-
C bonds present on biotin moieties. The N 1s spectrum, consisted of only one peak, centred at
400.3 eV, corresponds to amino (NH2) and amide (CONH) groups.
86
96
106
395400405Binding Energy (eV)
1:100 Biotin-2KC:TEGT mixed SAMs
6900
160164168Binding Energy (eV)
1:100 Biotin-2KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 96
Figure 4.13 – XPS spectra of N 1s (left) and S 2p (right) for biotin-6KC:TEGT 1:40 solution ratio
The XPS spectra for biotin-6KC:TEGT 1:2000 solution ratio are reported in Figure 4.14.
The S 2p spectrum presents two doublets peaks, the first one centred at 163.3 eV (S 2p1/2) and
161.8 eV (S 2p3/2), refers to the sulphur chemisorbed on the gold substrates, whereas the
second doublet peaks, centred at 163.6 eV and 165.0 eV, respectively, can be assigned to S-C
bonds present on biotin moieties. The N 1s spectrum presents an extremely small peak, that
was magnified, centred at 400.3 eV, can be assigned to amino (NH2) and amide (CONH) groups.
33
43
53
395400405Binding Energy (eV)
1:40 Biotin-6KC:TEGT mixed SAMs
18.5
160164168Binding Energy (eV)
1:40 Biotin-6KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 97
Figure 4.14 – XPS spectra of N 1s (left) and S 2p (right) for biotin-6KC:TEGT 1:2000 solution
ratio
The calculated surface ratios for both biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs
analysed are reported on Table 4.4.
Table 4.4 – Solution ratios of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs and relative
surface ratios calculated by XPS.
Ratio
Solution Surface
Biotin-2KC:TEGT 1:40 1:6 ± 1
1:100 1:16 ± 2
Biotin-6KC:TEGT 1:40 1:7 ± 2
1:2000 1:17 ± 2
From the results collected, it is possible to infer that the length of the oligopeptide
influences the surface ratio. To obtain a surface ratio of ≈1:5, a solution ratio of 1:40 is needed
21000
395400405Binding Energy (eV)
1:2000 Biotin-6KC:TEGT mixed SAMs
10100
160164168Binding Energy (eV)
1:2000 Biotin-6KC:TEGT mixed SAMs
N (1s) S (2p)
CHAPTER 4 98
for both biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs, whereas only a solution ratio of
1:10 was sufficient for the biotin-4KC:TEGT mixed SAMs. A different behaviour is observed for
the ≈1:16 surface ratio. In this case, the biotin-6KC:TEGT mixed SAMs need a higher content of
TEGT molecules in solution than the biotin-2KC:TEGT mixed SAM to obtain the same surface
ratio. This is due to a preferential absorption of the longer chain thiol. As stated before as the
chain length increases, the preferential adsorption of the longer thiol increases as
consequence.79,80,336,337
4.3.2 SPR analysis of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs switching properties
The switching properties of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAMs were
tested by electrochemical SPR, in the same ON (+0.3 V), OC and OFF (-0.4 V) conditions used
with biotin-4KC:TEGT and described before. The recorded SPR sensorgrams are reported in
Figure 4.15.
CHAPTER 4 99
Figure 4.15 – SPR sensorgram traces showing the binding of neutravidin to the biotin-
2KC:TEGT mixed SAMs (solution ratios of 1:40 and 1:100) and biotin-6KC:TEGT mixed SAMs
(solution ratios of 1:40 and 1:2000) under open circuit conditions, an applied positive (+0.3 V)
and negative(−0.4 V) potential.
The binding capacity and the switching efficiency of both biotin-2KC:TEGT and biotin-
6KC:TEGT mixed SAMs calculated from electrochemical SPR experiments are shown in Table
4.5.
CHAPTER 4 100
Table 4.5 – Binding capacity expressed in Resonance Units and switching efficiency
calculated from electrochemical SPR experiments on biotin-2KC:TEGT and biotin-6KC:TEGT
mixed SAMs.
Ratio
Binding Capacity
(RU)
Switching Efficiency
(%)
Solution Surface
Biotin-2KC:TEGT 1:40 1:6 ± 1 2634 ± 183 74 ± 4
1:100 1:16 ± 2 2295 ± 87 67 ± 8
Biotin-6KC:TEGT 1:40 1:7 ± 2 2822 ± 129 71 ± 7
1:2000 1:17 ± 2 229 ± 58 7 ± 4
By comparing the experimental data with those collected for biotin-4KC:TEGT mixed
SAMs at the same surface ratios, it is possible to see that the binding capacity of biotin-4KC and
biotin-2KC are similar, whereas it decreased significantly in the case of 1:16 biotin-6KC:TEGT
surface ratio. From the results obtained it is possible to assume that the low binding capacity
of biotin-6KC:TEGT mixed SAMs is due to the unfavourable orientation of the biotin molecules
on the surface, caused by the long and flexible six-lysine oligopeptide chains.
In the case of the ≈1:5 surface ratio, it is possible to observe a discrepancy between the
switching efficiency for the biotin-4KC:TEGT mixed SAM and the one for the biotin-2KC:TEGT
mixed SAM. This difference can be understood by looking at the calculated molecular lengths.
Biotin-4KC molecules measure 5.2 nm, needing a greater free volume on the surface than
biotin-2KC (3.4 nm), to successfully undergo the molecular switching. Notably, the switching
CHAPTER 4 101
efficiency of biotin-6KC:TEGT at the same surface ratio (≈1:5) is also higher than for the biotin-
4KC:TEGT mixed SAMs. This behaviour can be explained by considering the possible
intercrossing between biotin-6KC molecules, induced by conformation changes, reducing the
availability of biotin moieties for neutravidin binding. This can be due to the greater chain-chain
repulsion forces of longer charged oligopeptides, resulting in a highly-disordered arrangement
on the gold surface.339,340 In the case of ≈1:16 surface ratio, the switching of biotin-6KC:TEGT is
significantly impaired relative to biotin-2KC:TEGT and biotin-4KC:TEGT mixed monolayers. The
differences in switching efficiency demonstrated that the switching unit length plays and
important role in regulating the molecular changes triggered by the application of an electrical
potential. A longer switching unit can place constraints on the rearrangement of the biotin
moiety on the surface, such that it can be available or not for neutravidin binding.
4.2.5 Molecular Dynamics Simulations
The SPR results showed that the 1:40 biotin-4KC:TEGT solution ratio is the best
performing one, in terms of switching ability. When the biotin-4KC in the mixed SAM is high
(1:0-1:10 solution ratios), the oligopeptide backbone is unable to correctly undergo a molecular
rearrangement on the surface, resulting in the exposure of the biotin moiety also when a
negative potential is applied. As demonstrated by the SPR studies, a high concentration in
oligopeptide molecules in the surface results in a poor switching efficiency. The main
hypothesis arising from the observation made so far was that the ON/OFF behaviour of the
mixed biotin-4KC:TEGT, biotin-2KC:TEGT and biotin-6KC:TEGT SAMs is due to a spatial
modification of the switching backbone on the surface. This modification causes a change in
the availability of biotin on the gold substrate for binding with neutravidin in solution.
CHAPTER 4 102
To have an insight into the mechanism governing the switching performance of the
tested mixed SAMs, molecular dynamics (MD) simulations were performed by Dr Xingyong
Wang at Nanjing University (China). The MD simulations results are included in this dissertation
thesis to explain and support the electrochemical SPR results collected during this research
work. The success of MD simulations is strongly related to the force field used for the test.
Three different force fields were chosen and tested: cvff (consist-valence force field), compass
(condensed-phase optimized molecular potentials for atomistic simulation studies) and pcff
(polymer consistent force field). The cvff force field gave the best results and it was chosen for
our simulations. The surface models used in the simulations are shown in Figure 4.16.
Figure 4.16 – The surface models used in the MD simulations. The different colours in the
biotin-nKC chain represent the biotin moiety (purple), the four lysine (blue) and the cysteine
residues (dark green), respectively. Water molecules are represented by the orange dots. The
green and yellow balls denote the chloride ions and the gold atoms respectively, and the short
grey chains represent TEGT molecules.
CHAPTER 4 103
Two-dimensional rhombic periodic boundary condition and slab models were applied
during the simulations. The PBS buffer solution was simulated by employing water molecules
and chloride ions. The model parameters are summarised in detail in Chapter 3 – Experimental
Procedures and Protocols (Table 3.1).
The electrical potentials employed in the SPR experiments were modelled by applying
external electric fields. These electric fields caused a polarisation that can be considered by
employing the density functional theory-derived partial charge, in the simulations. Simulations
were carried out on biotin-nKC:TEGT (n= 2,4,6) and pure biotin-4KC SAMs.
An evident switching behaviour can be observed for biotin-2KC:TEGT (surface ratio 1:8)
and biotin-4KC:TEGT (surface ratio 1:16), when a positive electric field Ez was applied turning
the system to the “ON” state (Figure 4.17).
CHAPTER 4 104
Figure 4.17 – Conformational changes of biotin-2KC:TEGT (surface ratio of 1:8) and biotin-
4KC:TEGT (surface ratio of 1:16) mixed SAMs, under different electric field, along with the MD
simulation snapshots. The direction of the applied electric field is indicated by the black
arrows. Water molecules and hydrogen atoms are omitted for clarity. The gap distance
variation between the biotin moiety and the TEGT matrix is indicated by d.
In this state, the oligolysine chain is fully extended on the surface, completely exposing
the biotin head. Neutravidin molecules will then bind strongly with the available biotin moiety,
resulting in a high binding capacity, as verified by SPR. When a negative electric field E-z was
CHAPTER 4 105
applied, the system was switched to the “OFF” state. In this case, the oligopeptide backbone
rearranged on the surface, adopting a collapsed conformation. The biotin moiety resulted
being partially concealed by the TEGT molecules and unable to biointeract with neutravidin,
showing no bioactivity. When no electric field was applied, this corresponded to the OC state
studied by SPR. In this state, the switchable backbone adopted an intermediate conformation,
resulting in a moderate bioactivity. The MD simulation results were in fair agreement with the
data collected with the SPR experiments for the best-performing ratio of biotin-4KC:TEGT
mixed SAM. SPR analysis of the switching capacity revealed high binding when a positive
potential of +0.3V is applied.
The switching mechanism is controlled by the electrostatic interaction between the
positively charged oligolysine side chains and the applied electric field. In the case ofbiotin-
6KC:TEGT (1:15 surface ratio), the oligopeptide backbone is composed by six lysine, being too
long to allow the shielding of biotin moieties by TEGT molecules in the folded conformation
(Figure 4.18). Another important characteristic of note is that, when a negative electric field is
applied to the biotin-6KC:TEGT mixed SAM, the biotin head is still highly available for binding.
This can be observed from the gap distance variation between the biotin and the TEGT matrix
(d), which is more than 1.5 nm.
CHAPTER 4 106
Figure 4.18 – Conformational changes of biotin-6KC:TEGT (surface ratio of 1:15) mixed SAMs,
under different electric field, along with the MD simulation snapshots. The direction of the
applied electric field is indicated by the black arrows. Water molecules and hydrogen atoms
are omitted for clarity.
The MD simulations data were in agreement with the experimental data and confirmed
that a long switching unit is not suitable for controlling ligand bioactivity under an electrical
potential. These results are in line with previous research demonstrating that a longer biotin
linker lead to a high protein binding efficiency341.
X-ray crystallographic analysis showed that the biotin is buried quite deeply inside the
Neutravidin barrel, indicating that the biotin moiety needs to be completely inserted into the
Neutravidin binding pocket to have an efficient binding342–345. From these results, it is possible
to infer that a space of more than 1.5 nm is needed between the biotin moiety and the ethylene
glycol thiol matrix. These hypotheses are also consistent with the data obtained for biotin-2KC
and biotin-4KC under a negative electric field E-z and the poor binding capacity obtained in the
SPR experiments.
CHAPTER 4 107
In the case of OC conditions, the charged oligopeptide chains are not blocked in an
upright position by the positive potential, increasing the chances of intercrossing between the
oligopeptide chains, this phenomenon causes the biotin to be partially hindered from
interaction with biotin, justifying the lower bio-activity observed, compared to the ON state.
These findings can then be used to explain how each component of the mixed SAM and each
electrical condition govern the switching mechanism of the oligopeptide mixed monolayer.
The last consideration to be done is on the influence of the oligopeptide density on the
switching performance. In the case of pure biotin-4KC SAM, the oligopeptide chains are closely
packed on the surface, not allowing enough free space for the chains to undergo a molecular
rearrangement when a negative potential is applied (Figure 4.19).
Figure 4.19 – Conformational changes of the pure biotin-4KC SAM under different electric
fields (left) and MD simulation snapshots (right). L represents the variation of the gap distance
between the biotin end group and the gold substrate.
CHAPTER 4 108
Consequently, the biotin moieties were fully exposed on the surface at all time, leading
to a persistent bio-activity. This observation is consistent with the experimental data collected
by SPR in ON, OC and OFF conditions. It is therefore important to design mixed SAMs where
the switchable component possesses enough free space for conformational transitions.
4.4 Conclusions
Devices able to control the bioactivity of molecules on the surface, upon the application
of a stimulus, can have wide biological and medical applications in the study of cellular
processes regulation10,173. In this work, it was demonstrated that the switching mechanism of
charged oligopeptides mixed monolayers is based on a molecular rearrangement on the
surface, between a collapsed bio-inactive and a fully extended, bio-active conformation of the
oligopeptide chains.
These dynamic changes are controlled by the electrostatic attraction or repulsion
between the oligopeptide charged side chains and the substrate, resulting in a variation of the
gap distance (d) between the biotin end group and the ethylene glycol thiol matrix. When the
biotin moiety moves closer to the matrix, it is partially hindered and prevented from the
binding with neutravidin. On the contrary, when the oligopeptide chains are fully elongated on
the surface, the increase in the distance of the biotin from the matrix gives the biotin enough
free space to correctly be inserted in the Neutravidin binging pocket.
The detailed experimental and computational studies conducted in this research work,
imply that steric hindrance due to the neighbouring surface-confined oligopeptide chains,
significantly affects the switching efficiency of the mixed SAMs tested. The switching unit
length is an important factor to be considered in the design of switchable surfaces. In addition
CHAPTER 4 109
to this, the ratio of the SAMs component has to be optimised to maximise the switching
efficiency of the system.
The findings of our research can help in the development of new dynamic surface
materials that can find various applications in cellular studies and drug delivery systems.
CHAPTER 5 110
Chapter 5 - Study of the switching properties of progesterone-C7-4KC:EG6OH
mixed SAMs
Abstract: This chapter presents a detailed analysis of the development of switchable mixed
self-assembled monolayers exploiting the antigen-antibody (Ag-Ab) interaction. A model
system composed by a lysine oligopeptide carrying a progesterone moiety has been used for
the purposes of this work. Herein a detailed study is presented, on the feasibility of controlling
the interaction between the progesterone group and its anti-mouse antibody upon the
application of an electrical potential. The effect of different percentages of Bovine Serum
Albumin (BSA) on the switching abilities of the studied system has also been investigated.
Hexaethylene glycol thiol (EG6OH) molecules have been used as second component in the mixed
SAMs. The investigation has been conducted on different progesterone oligopeptide/EG6OH
ratios on the gold surfaces to identify the best performing one. The presence of EG6OH
molecules on the gold surface prevent any undesired antibody unspecific binding and donate
protein-resistant characteristics to the surface itself. In addition, it provides molecular space to
the oligopeptide units to efficiently undergo their molecular rearrangement on the gold surface,
upon the application of an electrical potential.
5.1 Introduction
The interaction between an antigen and its relative antibody has been widely exploited
in several immunosensors platforms346. Such biosensors exploit the biorecognition between a
ligand on the surface (usually the antibody) and its binding partner in solution, called analyte
(antigen)347. These devices find important application in biomedical research, for diagnostic
purposes, especially for the identification of cancer markers. In this case, the ability to detect
low concentration of biomolecules involved in the early stages of cancer development is of
CHAPTER 5 111
paramount importance for the success of cancer therapies and still remains one of the major
challenges for researchers348.
A well-known, label-free detection method for immunosensing is provided by surface
plasmon resonance349. SPR was used for immunosensing in 1983 for the first time283, since then
several SPR platform exploiting the antibody-antigen interaction have been developed. These
platforms usually present the antibody immobilised on the metal surface, that is able to
discriminate between different isoforms of an antigen in solution. However, due to the small
molecular weight of the antigen molecules, if compared to the antibody one, the SPR response
could not always be detectable347. The immobilisation of an antigen chemically modified with
a molecular linker to allow the binding to the metal surface present the advantage of enhancing
the surface plasmon resonance response when a high-molecular weight antibody, leading to a
large shift in the SPR signal347.
The current research on immunosensors does not present any work on the control of
the antigen exposure on the sensor surface upon the application of an electrical potential. The
interaction is always studied in static conditions and it is only used to either discriminate
between different antigens able to bind to the same antibody or between different antibodies,
able to bind the same epitope on an antigen molecules350,351. Designing a smart surface able to
expose or conceal an antigen molecule, by simply switching between a positive and a negative
potential can easily find an application in the biomedical research field.
In Chapter 4 we described how to selectively control the interaction between a biotin
moiety bound to a 4-lysine chain and anchored to the gold surface thanks to a cysteine
molecule and molecules of neutravidin in solution. The same system was also used to
investigate the concealment and exposure of arginine–glycine–aspartate (RGD) oligopeptides
CHAPTER 5 112
to control cell adhesion, with successful results199. Starting from this background, the antigen
molecules can be chemically modified by binding lysine oligopeptide chains that have already
been successfully used as switching units196–199. The lysine aminoacid presents a positive charge
at pH 7, that can be exploited to obtain a molecular rearrangement of defined molecules on
the sensor surface.
In this work, we want to develop a novel system able to control the exposure of a
chemically modified antigen anchored to the gold surface, by applying an electrical potential.
In particular, we want to achieve the possibility, to control the exposure of progesterone
moieties on the sensor surface, never analysed before. The results of this work can then be
used to investigate the hyperactivation of sperm cells, one of the crucial steps during
fertilisation238,239.
In this work, different mixed SAM were studied, with molecules of progesterone as the
antigen end group. The progesterone molecule was bound to an alkyl chain through an oxime
group, connected to a switchable oligolysine chain, used in our previous work197. The first
component of the chosen mixed SAM for this work, is a lysine (K) oligopeptide, functionalised
at one end with a progesterone moiety, able to strongly interact with the anti-mouse
progesterone antibody, and at the other end with an aminoacid of cysteine (C) to anchor the
oligopeptide to the gold substrates via the thiol group. The alkyl chain composed by eight
methylene groups has been chosen to space out the progesterone moiety from the hydrophilic
chain of four lysine. A hydrophobic spacer will allow the progesterone to be inserted inside the
sperm cells hydrophobic membrane, where its receptors are located352–355. The second
component is represented by a hexaethylene glycol-terminated thiol (EG6OH). The aim of
EG6OH molecules is both to space out the oligopeptide to allow enough space on the surface
CHAPTER 5 113
for molecular rearrangement and impede any undesired unspecific binding of anti-mouse
progesterone antibody on the gold surfaces (Figure 5.1).
Figure 4.1 - Molecular structures and related cartoons of the oligopeptide (progesterone-C7-
4KC) and the hexaethylene glycol-terminated thiol (EG6OH) used in the mixed SAMs, and their
calculated molecular lengths in fully extended conformations.
The results of this analysis will permit a better design of novel sensing platforms, that
can discriminate between damaged and healthy sperm cells, applicable to in-vitro Fertilisation
(IVF) research purposes.
CHAPTER 5 114
5.2 Objectives
1. Characterisation of different progesterone-C7-4KC:EG6OH solution ratios (1:10, 1:40
and 1:100) on gold surfaces by XPS, to evaluate the differences between solution ratio
and surface ratio.
2. Analysis, by electrochemical SPR of the specificity of the anti-mouse progesterone
antibody, in OC conditions, on both a pure biotin-4KC and a pure EG6OH SAMs.
3. Analysis, by electrochemical SPR, of the switching properties in PBS of the different
mixed SAM ratios, to select the one with the highest switching efficiency and develop a
novel platform for the control over the antigen-antibody interaction, upon the
application of an electrical potential. The switching efficiency of the best performing
mixed SAM will be then be tested in sEBSS+0.3%BSA, buffer that will be used for the
preparation of sperm cells (see Chapter 6).
4. Comparison of the best performing surface ratio of progesterone-C7-4KC switching
efficiency in PBS, EBSS and EBSS containing different percentages of BSA (0.1% and
0.3%)
CHAPTER 5 115
5.3 Results and Discussion
5.3.1 Formation of mixed progesterone-C7-4KC:EG6OH mixed SAMs
The aim of this work is to develop a switchable sensing platform able to control the
exposure of progesterone molecules on the gold substrates. Piranha-cleaned gold substrates
were incubated into different solution ratios of mixed progesterone-C7-4KC:EG6OH SAMs.
In this research work, we chose to immobilise a molecule of progesterone (antigen)
purposely chemically modified to contain a switching unit and a sulphur atom for the binding
to the gold surface. This strategy will allow the obtainment of an enhanced SPR response when
the antibody in solution will bind to the progesterone moiety anchored to the gold surface347.
When a negative potential is applied to the to the gold substrate, the lysine oligopeptide
is expected to be attracted towards the surface. This attraction will drag the progesterone
moiety, that will then be hindered from the interaction with the anti-mouse monoclonal
progesterone antibody, by the EG6OH molecules. On the contrary, when a positive potential is
applied the lysine oligopeptide will be repelled by the positive charge on the gold surface, being
fully extended on the surface, therefore completely exposing the progesterone moiety for the
interaction with the monoclonal anti-mouse antibody. EG6OH molecules were employed to
prevent non-specific binding of antibody on the surface and to give enough conformational
freedom to the progesterone-C7-4KC component to undergo the switching (Figure 5.2).
CHAPTER 5 116
Figure 5.2 - Cartoon representation of the ON-OFF switching system that controls the
biomolecular interaction between progesterone (red) on the surface and antibody (green) in
solution.
5.3.2 XPS characterisation of progesterone-C7-4KC:EG6OH mixed SAMs
One of the aims of this research work is to create a mixed SAM platform that can present
a similar switching ability to the one obtained with the biotin-4KC:TEGT system, described in
Chapter 3197. Three progesterone-C7-4KC:EG6OH solution ratios of 1:10, 1:40 and 1:100 were
characterised by XPS. The 1:40 solution ratio was chosen, because, as demonstrated in Chapter
3, it gives to the switching oligopeptide the correct spatial area on the surface to undergo the
desired molecular rearrangement197.
5.3.2.1 Progesterone-C7-4KC:EG6OH 1:10 solution ratio
The XPS analysis carried out for the 1:10 mixed SAM revealed the presence of the
elemental species S, N, C and O, confirming the formation of the mixed SAM. The S 2p spectrum
(Figure 5.3 a), consists of a doublet peak at 163.2 eV (S 2p1/2) and 162.1 eV (S 2p3/2)
corresponding to the S-Au bond252,255,334,335; the second doublet peak centred at 164.7 (S 2p1/2)
CHAPTER 5 117
eV and 163.8 eV (S 2p3/2) can be assigned to the S-H group, due to the presence of an undesired
amount of free, unbound thiol molecules on the gold surface252,255,334,335. The unattached
molecules can be either those of the ethylene glycol thiol or those of the oligopeptide
molecules.
In the N 1s spectrum (Figure 5.3 b), only one peak can be fit, due to the high background
noise. This peak is centred at 400.0 eV, attributable to amino (NH2) and amide (CONH) groups
and one 252,255,334,335.
The C 1s spectrum can be de-convoluted into three peaks (Figure 4.3 c), attributable to
five different binding environments. The first peak at 284.8 eV corresponds to C-C bonds, the
second peak at 286.6 eV can be attributed to the binding environments of C-S, C-N and C-O and
the smaller third peak at 288.0 eV corresponds to the carbonyl moiety C=O252,255,334,335.
The O 1s spectrum (Figure 4.3 d) can be de-convoluted into two different peaks, one at
532.7 eV corresponding to C-O bonds and one at 531.3 eV corresponding to C=O
bonds252,255,334,335.
CHAPTER 5 118
Figure 5.3 – XPS spectra of the a) S 2p, b) N 1s, c) C 1s and d) O 1s regions for the 1:10
progesterone-C7-4KC:EG6OH mixed SAM solution ratio.
To calculate the surface ratio of the mixed SAM, the areas of S 2p and N 1s peaks were
integrated and it was taken into consideration that the progesterone-C7-4KC oligopeptides
(Figure 5.1) contain 11 N atoms and 1 S atom and that EG6OH presents no N atoms and only 1
S atom. Equation 5.1 was used to calculate the number of ethylene glycol thiol molecules per
oligopeptide on the gold surface.
CHAPTER 5 119
𝑁𝑜. 𝑜𝑓𝐸𝐺6𝑂𝐻 = (𝑁𝑜. 𝑜𝑓𝑁𝑝𝑒𝑟𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑥𝑆𝑎𝑟𝑒𝑎
𝑁𝑎𝑟𝑒𝑎) − 𝑁𝑜. 𝑜𝑓𝑆𝑝𝑒𝑟𝑝𝑒𝑝𝑡𝑖𝑑𝑒
Equation 5.1
The calculated surface ratio for the progesterone-C7-4KC:EG6OH mixed SAM,
corresponded to 1:9±3, different from the solution ratios, as described in previous studies of
two-components SAMs79,80.
The surface ratio obtained for the 1:10 progesterone-C7-4KC:EG6OH mixed SAMs is
slightly higher compared to the one obtained for the 1:10 biotin-4KC:TEGT mixed SAMs solution
ratio. This discrepancy could be due to the longer chains composing the progesterone-C7-
4KC:EG6OH mixed SAMs. In addition, the presence of different chemical groups attached to
the oligopeptide chains and the higher number of ethylene glycol thiol groups in the EG6OH
molecules, compared to the TEGT molecules can drive to a different chemical rearrangement
of thiols on the gold surfaces35,87,108,356,357. By observing the behaviour of longer oligopeptide
molecules of Biotin-6KC, analysed in Chapter 4, it is possible to hypothesise that the high
concentration of progesterone-C7-4KC molecules will lead to a similar molecular intercrossing
phenomenon, resulting in a poor switching efficiency of the 1:10 solution ratio. This hypothesis
will be verified by electrochemical SPR in section 5.4.2.
5.3.2.2 Progesterone-C7-4KC:EG6OH 1:40 solution ratio
In Chapter 3 was shown that 1:40 solution ratio of Biotin-4KC:TEGT mixed SAM,
corresponding to 1:16±4 surface ratio, possess the best switching characteristics amongst the
system tested. Thus, the same solution ratio was first tested and characterised, to analyse how
the differences between the biotin-4KC:TEGT mixed SAMs and the
CHAPTER 5 120
progesterone-C7-4KC:EG6OH mixed SAMs, such as the molecular length of the SAMs
components, could influence their organisation on the gold surface.
XPS analysis showed the presence of S, N, C and O elements (Figure 5.4) confirming the
formation of the mixed SAM. The S 2p spectrum (Figure 5.4 a) consists of two doublet peaks,
the first one at 163.3 eV (S 2p1/2) and 162.1 eV (S 2p3/2), assignable to the sulphur chemisorbed
to the gold surface252,255,334,335. The second one is centred at 163.8 eV (S 2p3/2) and 165 eV (S
2p1/2) assignable to unbound sulphur present on the gold surface252,255,334,335. The N 1s
spectrum (Figure 5.4 b) is de-convoluted into one single peak, centred at 399.7 eV
corresponding to amino (NH2) and amide (CONH) groups252,255,334,335. As expected, the N 1s
spectrum is smaller than the one recorded for the 1:10 progesterone-C7-4KC:EG6OH mixed
SAM ratio.
The C 1s spectrum (Figure 5.4 c) is de-convoluted into three peaks, which can be
attributed to five different binding environments. The peak at 284.7 eV corresponds to C-C
bonds, while the peak at 286.6 eV corresponds to the three binding environments of C-S, C-N
and C-O. The smaller peak at 287.7 eV can be assigned to the carbonyl moiety C=O252,255,334,335.
The O 1s spectrum (Figure 5.4 d) is de-convoluted into two peaks, the first one at 533.1 eV is
attributable to C-O bonds and the second one at 531.8 eV corresponds to C=O
bonds252,255,334,335.
CHAPTER 5 121
Figure 5.4 – XPS spectra of the a) S 2p, b) N 1s, c) C 1s and d) O 1s regions for the 1:40
progesterone-C7-4KC:EG6OH mixed SAM solution ratio.
By using Equation 5.1 it is possible to calculate the surface ratio for the progesterone-
C7-4KC:EG6OH mixed SAM, corresponding to 1:22±5, not matching the solution ratio, as
expected79,80. The differences with the surface ratio obtained from the 1:40 biotin-4KC solution
ratio can be attributed to the different chain lengths of the components of the mixed
monolayer, the different end group on the oligopeptide chain and the different type of
ethylene glycol thiol. In fact, EG6OH molecules are composed by six ethylene glycol groups and
a spacer of 11 carbon atoms, between the ethylene glycol groups and the sulphur head group,
offering the conditions to form a highly-packed matrix35,87,108,356,357.
CHAPTER 5 122
5.3.2.3 Progesterone-C7-4KC:EG6OH 1:100 solution ratio
The XPS analysis of the Progesterone-C7-4KC:EG6OH mixed SAM at a solution ratio of
1:100 showed the presence of S, C and O elements, but no N signal could be recorded (Figure
5.5).
The S 2p spectrum (Figure 5.5 a) present two doublet peaks, the first one centred at
163.3 eV (S 2p1/2) and 162.1 eV (S 2p3/2), that can be attributed to the sulphur atom of the thiol
groups, covalently bound to the gold substrate252,255,334,335. The second one is visible at 164.8
eV (S 2p1/2) and 163.7 eV (S 2p3/2) and it is ascribable to the unbound sulphur (S-H) present on
the gold surface252,255,334,335. However, it is possible to infer that a small quantity of unbound
sulphur is present on the gold surface. The N 1s spectrum was recorded (Figure 5.5 b) but no
peak could be identified, due to high noise of the background, not allowing a quantitative
interpretation of the surface ratio. The C 1s spectrum (Figure 5.5 c) showed the presence of
five different binding environments and it can be de-convoluted into three peaks. The first one
at 284.8 eV is referred to C-C bonds, the second peak at 286.6 eV can be assigned to C-S, C-N
and C-O bonds. The smaller peak at 288.0 eV is assigned to the carbonyl moiety
(C=O)252,255,334,335.
The O 1s spectrum (Figure 5.5 d) can be de-convoluted into two peaks, corresponding to the
binding environments of C-O (532.8 eV) and C=O (531.3 eV), respectively252,255,334,335.
CHAPTER 5 123
Figure 5.5 – XPS spectra of the a) S 2p, b) N 1s, c) C 1s and d) O 1s regions for the 1:100
progesterone-C7-4KC:EG6OH mixed SAM solution ratio.
Due to the impossibility of recording the nitrogen spectrum, it is possible to hypothesise
a high percentage of ethylene glycol thiol molecules on the gold surface, forming a well-packed
EG6OH matrix. This matrix should confer slightly hydrophilic characteristics to the 1:100
solution ratio surfaces and also constrain the oligopeptide molecules from undergoing a
molecular rearrangement over the surface, leading to a low switching ability.
Ellipsometry, contact angle and SPR analysis of the selected surfaces were conducted
to verify our hypothesis and are illustrated in the next sections.
CHAPTER 5 124
5.3.3 Contact angle and ellipsometry characterisation of progesterone-C7-4KC:EG6OH mixed
SAMs
The formation of progesterone-C7-4KC:EG6OH mixed monolayers, at 1:10; 1:40 and
1:100 solution ratios, were analysed by contact angle and ellipsometry. Pure progesterone-C7-
4KC and pure EG6OH SAMs were characterised as a control (Table 4.1). The water advancing
(Adv) and receding (Rec) contact angles for EG6OH revealed hydrophilic characteristics,
whereas the pure progesterone-C7-4KC presented more hydrophobic characteristic. This
behaviour can be due both to the oligopeptide molecules being collapsed on the chip surface,
exposing the hydrophobic 8-carbon alkyl chain, and to the hydrophobic progesterone end
group.
Ellipsometry analysis confirmed the formation of pure progesterone-C7-4KC SAMs, pure
EG6OH SAM and mixed progesterone-C7-4KC:EG6OH mixed SAMs, but, as expected, the
homogeneity of the mixed monolayers on surface was poor, due to difficulties in controlling
the organisation of the two components of the mixed SAM on the gold surfaces. Ellipsometry
results were fitted using a model based on the Cauchy equation, an empirical relationship
between the refractive index and wavelength of light, which considers a SAM as a transparent
layer. The thickness of the SAM was then calculated using multi-guess iterations that provide a
thickness result with the lowest χ2 (chi-square distribution) between the measured and the
calculated values of ψ and Δ (see section 2.3). The formation of pure progesterone-C7-4KC
monolayer resulted in an ellipsometric thickness of 4.4 ± 0.2 nm which is significantly lower
that the calculated theoretical molecular length of 6 nm. As mentioned above, this difference
can be explained with the oligopeptide chains being collapsed on the surface, exposing the
hydrophobic alkyl spacers. The exact theoretical molecular length of progesterone-C7-
4KC:EG6OH mixed SAM cannot be calculated, because this monolayer is composed by two
CHAPTER 5 125
molecules presenting different molecular length. However, it is important to notice that the
molecular lengths measured for the different ratios of the mixed SAM resulted in between the
measured lengths for the pure progesterone-C7-4KC SAM and the EG6OH SAM. Interestingly,
as the concentration of ethylene glycol molecules increased in the mixed SAM solution, the
measured length became closer to the pure EG6OH SAM measure length, confirming the
increment in the number of ethylene glycol thiol molecules on the surface.
Table 5.1 - Advancing and receding water contact angle and ellipsometric thickness for the
different monolayers formed for 24h. The calculated molecular lengths were determined using
ChemBio3D Ultra 12.0 in which the molecules were in fully extended conformation. The
averages and standard errors were calculated from at least three different measurements.
Layer Contact Angle Thickness (nm)
Adv. Rec. Calc. Measured
Progesterone-C7-4KC 0.1mM 70° ± 3 67° ± 4 6 4.4 ± 0.2
EG6OH 0.1 mM 41° ± 2 39° ± 3 3.3 2.4 ± 0.1
Progesterone-C7-4KC:EG6OH
1:10
52° ± 2 45° ± 3 - 3.8 ± 0.5
Progesterone-C7-4KC:EG6OH
1:40
43° ± 2 38° ± 4 - 3.1 ± 0.4
Progesterone-C7-4KC:EG6OH
1:100
40° ± 5 35° ± 2 - 2.8 ± 0.3
CHAPTER 5 126
The contact angle results indicate that progesterone-C7-4KC:EG6OH mixed SAMs
presented hydrophilic characteristics, comparable to the one obtained for the pure ethylene
glycol thiol monolayer, indicating the larger presence of this component on the gold surface,
compared to the oligopeptide chains. It has to be highlighted that, as the concentration of the
EG6OH component increased on the gold surface, the mixed SAMs acquired more hydrophilic
characteristics. The large hysteresis (θAdv – θRec) presented by the mixed progesterone-C7-
4KC:EG6OH monolayers are an indication of the low order of both layers. In addition, the
difference in molecular length of the two SAM components lead to an uneven surface, causing
such a large difference between the advancing and receding angles.
The measured thickness of both the pure progesterone-C7-4CK and pure EG6OH SAMs
are lower than the theoretical thickness calculated using ChemDraw 3D. It is possible to infer
that on both the resulting monolayer the molecules are partially collapsed on the gold surface,
leading to a smaller thickness than the expected one.
5.4 SPR analysis of the progesterone-C7-4KC:EG6OH mixed SAMs and anti-mouse
progesterone antibody
5.4.1 Testing the anti-mouse progesterone antibody specificity by SPR
Before starting the analysis of the switching capability of the chosen system, it was
necessary to verify that the anti-mouse progesterone antibody was not recognising the second
component of the mixed monolayer, EG6OH, as possible epitope. Pure EG6OH SAMs were
tested by electrochemical SPR in OC conditions, in the presence of the antibody. A biotin-
4KC:EG6OH mixed SAM of 1:40 solution ratio was also tested as an additional control.
CHAPTER 5 127
Phosphate buffer saline (PBS) was flushed over the surface for 10 minutes to equilibrate
the sensor chip and set the SPR baseline, in OC (no potential) state. This step was followed by
injection of monoclonal anti-mouse progesterone antibody, diluted in degassed PBS, over the
surface for 30 minutes, necessary to potentially allow the correct interaction between the
antibody and both EG6OH and biotin molecules, attached to the gold substrate. After this
exposure time, the chip was rinsed with degassed PBS, to remove any unbound protein still
present in the sensor cell and the binding capacity was recorded.
As expected, no significant SPR signal shift was recorded with both EG6OH and
biotin-4KC:EG6OH SAMs (Figure 5.6).
Figure 5.6 - SPR sensorgram, recorded in OC (no potential applied) conditions, for the injection
of anti-mouse progesterone antibody on both biotin-4KC:EG6OH and EG6OH SAMs.
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The results obtained confirmed that the second component of the
progesterone-C7-4KC:EG6OH mixed SAM could not affect the binding capacity of the antibody
to its relative antigen on the surface (progesterone moieties).
5.4.2 SPR analysis of the switching capabilities of progesterone-C7-4KC:EG6OH mixed SAMs
in Phosphate Saline Buffer
To understand if the progesterone-C7-4KC:EG6OH mixed SAMs could control the
progesterone exposure upon the application of an electrical stimulus, the three different
solution ratios, 1:10, 1:40 and 1:100 and previously analysed by XPS, were tested by
electrochemical SPR in Phosphate-Buffered Saline (PBS). These preliminary SPR experiment
had the aim of identifying the best performing ratio to be selected to be tested in sEBSS+0.3%
BSA. Switching studies were conducted by monitoring the binding of the monoclonal anti-
mouse progesterone antibody to the progesterone end groups on the oligopeptide. Our
research group had already demonstrated that oligopeptide mixed SAM can be used to control
both protein adhesion and cell adhesion196, upon the application of an electrical potential,
without affecting the SAM integrity, as also illustrated in Chapter 4. Thus, the same electrical
potentials were used in this research work. The SPR experiments were conducted in three
different states: bio-active (ON), while a positive potential of +0.3 V was applied, open circuit
(OC) while no potential was applied and bio-inactive (OFF), while a negative potential of -0.4 V
was applied. The recorded sensorgrams are reported in Figure 5.7.
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Figure 5.7 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at a) 1:10, b) 1:40 and c) 1:100 solution ratio, in PBS, under OC
(no applied potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
PBS was flushed over the surface for 10 minutes to equilibrate the sensor chip and set
the SPR baseline, in either ON (+0.3 V), OC (no potential) or OFF (-0.4 V) states. This step was
followed by injection of monoclonal anti-mouse progesterone antibody, diluted in degassed
PBS, over the surface for 30 minutes, necessary to allow the correct interaction between the
antibody and its antigen, represented by the progesterone moieties anchored to the gold
substrate. After this exposure time, the chip was rinsed with degassed PBS, to remove any
unbound protein still present in the sensor cell and the binding capacity was recorded. The
binding capacity (BC) is defined as the difference in the SPR response units between the
beginning of the antibody injection and the end of washing with PBS. The averages and the
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standard errors in the reported binding capacity were calculated from at least three different
SPR measurements for each ratio (Table 5.2).
Table 5.2 – Progesterone-C7-4KC:EG6OH Binding Capacity (BC), expressed in Resonance Units
(RU) and Switching Efficiency calculated from SPR experiments.
Progesterone-C7-4KC:EG6OH
Ratio Binding Capacity (RU) Switching Efficiency (%)
Solution Surface
1:10 1:9±3 1173±35 25±6
1:40 1:22±5 1195±22 73±3
1:100 - 526±149 37±17
The progesterone-C7-4KC:EG6OH mixed SAMs at solution ratio of 1:10 and 1:40 show
high antibody binding with immobilization capacities of 1.17 ng/mm2 and 1.20 ng/mm2,
respectively (1000 RU = 1 ng/mm2).196 However, for the solution ratio at 1:100, there is a
significant decrease in antibody binding (0.53 ng/mm2) that reflects a decrease in the amount
of progesterone-C7-4KC in the mixed SAM. This behaviour is to a certain extent expected and
indicates that there is good accessibility of the antibody to the surface tethered-antigen at
progesterone-C7-4KC:EG6OH surface ratios of 1:9 and 1:22. The switching efficiency (SE) was
calculated as the percentage between the binding capacity when a positive potential was
applied (BCON) and the binding capacity when a negative potential was applied (BCOFF), divided
by BCON:
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𝑆𝐸 =𝐵𝐶𝑂𝑁−𝐵𝐶𝑂𝐹𝐹
𝐵𝐶𝑂𝑁𝑥100
Equation 5.2
From the results obtained, it is possible to infer that both 1:10 and 1:100 progesterone-
C7-4KC:EG6OH SAMs cannot provide enough control over the anti-mouse progesterone
antibody over the gold surface, showing only 25±6% and 37±17% of switching efficiency,
respectively (Figure 5.8).
Figure 5.8 – Bar Chart representing the switching efficiency obtained for 1:10, 1:40 and 1:100
progesterone-C7-4KC:EG6OH mixed SAMs solution ratios
In the case of the 1:10 solution ratio, the results can be attributed to a high density of
progesterone-C7-4KC chains. These long switching chains can cause intercrossing between
each other, with a similar phenomenon as the one seen in Chapter 4 for biotin-6KC molecules.
This behaviour is confirmed by the low binding capacity recorded in the OC conditions, that
resulted lower than the binding capacity when a negative potential was applied (Figure 5.7 a).
CHAPTER 5 132
In this case, the oligopeptide molecules are not forced by the electrical potential to be oriented
in a particular direction, therefore they present a higher flexibility that can lead to an increased
molecular intercrossing occurrence. It must be highlighted that the 1:10 solution ratio presents
a higher binding capacity, but a lower switching efficiency, if compared to the one obtained for
the 1:40. The results can be attribute to the high concentration of EG6OH molecules on the
gold substrate. The highly-packed matrix of ethylene glycol thiol molecule can force the
progesterone-C7-4KC molecules in an upright orientation, not leaving free movement to the
alkyl spacer to possibly orient the progesterone moiety in the wrong direction for the binding
to the antibody, as it could happen in the case 1:40 solution ratio where the oligopeptide
molecules are more spaced out.
In the case of the 1:100 progesterone-C7-4KC:EG6OH solution ratio, the decrease in the
binding capacity of the mixed SAM is due to the reduction of progesterone presence on the
surface. The phenomena mentioned above resulted in poor switching capabilities of both 1:10
and 1:100 mixed SAMs ratios. In addition, as observed in the case of biotin-4KC:TEGT mixed
SAMs, the decrease in the binding capacity is not proportional with the reduction of the
concentration of biotinylated oligopeptide on the surface. Again, it is possible to assume that
both the steric hindrance and the restricted mobility of the densely-packed oligopeptide
molecules might limit the antibody binding to the progesterone moiety at low ratio of EG6OH
to progesterone-C7-4KC197. The data collected confirmed the phenomenon observed in
Chapter 4: a densely-packed switchable oligopeptide SAM cannot undergo a molecular
rearrangement on the surface and some free space is needed for the oligopeptide chains to
change their conformation when a negative potential is applied.
CHAPTER 5 133
Noticeably, the switching efficiency obtained for the 1:40 progesterone-C7-4KC:EG6OH
solution ratio is much lower than the one for the same solution ratio of biotin-4KC:TEGT. The
difference in the switching behaviour can either be explained by the different surface ratio
and/or the different chemical composition of the switching molecules. In fact, in the case of
the 1:40 progesterone-C7-4KC:EG6OH solution ratio, the resulting surface ratio is higher than
the biotin-4KC:TEGT one (1:22±5 vs 1:16±4). In addition, the progesterone molecule is not
directly bound to the oligolysine chain and the alkyl spacer is not affected by the application of
an electrical potential. This chemical characteristic can lead to lower constraints affecting the
exposure of the progesterone molecules upon the application of a negative electrical potential
and it can donate more free movement to the progesterone moieties for the binding to the
antibody in solution.
The 1:22 surface ratio (1:40 solution ratio) of the progesterone-C7-4KC:EG6OH mixed
monolayer presented the highest switching efficiency and was selected to study the switching
efficiency of the progesterone oligopeptide in sEBSS containing 0.4% BSA.
5.4.3 SPR analysis of the switching capabilities of 1:40 progesterone-C7-4KC:EG6OH mixed
SAMs solution ratio in modified Earle’s Buffer Saline Solution containing 0.3% of Bovine
Serum Albumin
To understand if the best performing progesterone-C7-4KC:EG6OH mixed SAMs ratio
could present a similar switching efficiency in the buffer that will be used for the treatment of
sperm cells, the electrochemical SPR experiments illustrated in section 5.4.2, were repeated in
sEBSS + 0.3% BSA. The sEBSS buffer mimics the uterus environment, whereas the BSA is
necessary for the sperm cells to lose the cholesterol external layer in order to undergo
CHAPTER 5 134
capacitation and help their motility223,236,242,358–360. It is therefore important to verify if the
molecular rearrangement over the surface can be induced also in the sEBSS+0.3% BSA buffer.
The same procedure illustrated in section 5.4.2 was followed, monitoring the binding of the
monoclonal anti-mouse progesterone antibody to the progesterone end groups on the
oligopeptide. The SPR experiments were conducted in three different states: bio-active (ON),
while a positive potential of +0.3 V was applied, open circuit (OC) while no potential was applied
and bio-inactive (OFF), while a negative potential of -0.4 V was applied.
Modified Earle’s Buffer Saline Solution (sEBSS) containing 0.3% of BSA was flushed over
the surface for 10 minutes to equilibrate the sensor chip and set the SPR baseline, in either ON
(+0.3 V), OC (no potential) or OFF (-0.4 V) states. This step was followed by injection of
monoclonal anti-mouse progesterone antibody, diluted in sEBSS+0.3%BSA, over the surface for
30 minutes, necessary to allow the correct interaction between the antibody and its antigen,
represented by the progesterone moieties anchored to the gold substrate. The buffer was not
degassed, due to the presence of BSA that could have caused the formation of bubbles, under
the argon flux. In addition, the sperm cells will be prepared using not degassed
sEBSS+0.3%BSA312. After this exposure time, the chip was rinsed with sEBSS + 0.3% BSA, to
remove any unbound antibody still present in the sensor cell and the binding capacity was
recorded. The recorded sensorgram is reported in Figure 5.9.
CHAPTER 5 135
Figure 5.9 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at 1:40 solution ratio, in sEBSS + 0.3% BSA, under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
The binding capacity (BC) and the switching efficiency (SE %) in sEBSS + 0.3% BSA were
calculated using Equation 5.2, to obtain values of 1048±286 and 35±10%. The averages and the
standard errors in the reported binding capacity were calculated from at least three different
SPR measurements. The results showed that the switching efficiency in sEBSS + 0.3% BSA is
much lower compared to the one obtained in PBS and it is too poor to permit the achievement
of an efficient switching system in the presence of sperm cells. The difference in the switching
performances can be attributed to the presence of Bovine Serum Albumin. In fact, BSA is a non-
reactive protein used in immunohistochemistry to bind to non-specific binding sites, to
increase the chances for the antibodies to bind only to the specific antigens361,362. In addition,
BSA also present an overall negative charge at pH 7363. From the analysis of these two
characteristics, we can infer that BSA could be interacting with either the positively-charged
CHAPTER 5 136
oligolysine peptide chains and/or the negatively-charged gold surface, when the -0.4 V
potential is applied364, leading to a poor switching behaviour and a high standard error.
5.4.4 SPR analysis of the switching capabilities of 1:40 progesterone-C7-4KC:EG6OH mixed
SAMs solution ratio in modified Earle’s Buffer Saline Solution containing 0.1% and 0% of
Bovine Serum Albumin
To understand if the presence of BSA could interfere with the switching as hypothesise
in the previous section, electrochemical SPR switching experiments were conducted on the
1:40 progesterone-C7-4KC:EG6OH mixed SAMs solution ratio, in both sEBSS+0.1%BSA and
sEBSS+0%BSA as buffer. This detailed analysis will give us an in-depth understanding of the
molecular events occurring on the gold surface, that will be also be fundamental to plan our
future studies.
Again, the procedure used in section 5.4.2 was followed, monitoring the binding of the
monoclonal anti-mouse progesterone antibody to the progesterone end groups on the
oligopeptide. The SPR experiments were conducted in three different states: bio-active (ON),
while a positive potential of +0.3 V was applied, open circuit (OC) while no potential was applied
and bio-inactive (OFF), while a negative potential of -0.4 V was applied.
Firstly, modified Earle’s Buffer Saline Solution (sEBSS) containing 0.1% of BSA was
flushed over the surface for 10 minutes to equilibrate the sensor chip and set the SPR baseline,
in either ON (+0.3 V), OC (no potential) or OFF (-0.4 V) states. This step was followed by injection
of monoclonal anti-mouse progesterone antibody, diluted in sEBSS+0.3%BSA, over the surface
for 30 minutes, necessary to allow the correct interaction between the antibody and its
antigen, represented by the progesterone moieties anchored to the gold substrate. The buffer
CHAPTER 5 137
was not degassed for the same reasons explained in the previous section312. After this exposure
time, the chip was rinsed with sEBSS + 0.1% BSA, to remove any unbound antibody still present
in the sensor cell and the binding capacity was recorded. The recorded sensorgram is reported
in Figure 5.10.
Figure 5.10 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at 1:40 solution ratio, in sEBSS + 0.1% BSA, under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
The SPR analysis in sEBSS + 0.1% BSA showed that the reduced amount of Bovine Serum
Albumin slightly improved the binding efficiency from the value of 1048±286 obtained in sEBSS
+ 0.3% BSA to 1291±78. The switching efficiency, calculate using Equation 4.2, resulted
increased to 44±8%. However, the switching efficiency remains too poor to select the buffer
sEBSS + 0.1% BSA as ideal to perform the switching experiments in the presence of sperm cells.
It is also important to notice that in the presence of BSA the standard error for the calculated
CHAPTER 5 138
switching efficiency is high, indicating that the protein is critically affecting the possibility of
obtaining the desired molecular rearrangement on the gold surfaces, upon the application of
an electrical potential.
Finally, we analysed the switching behaviour of the progesterone-C7-4KC:EG6OH mixed
SAM in presence of no BSA in the sEBSS buffer. Modified Earle’s Buffer Saline Solution (sEBSS)
containing 0.1% of BSA was flushed over the surface for 10 minutes to equilibrate the sensor
chip and set the SPR baseline, in either ON (+0.3 V), OC (no potential) or OFF (-0.4 V) states.
This step was followed by injection of monoclonal anti-mouse progesterone antibody, diluted
in sEBSS+0.3%BSA, over the surface for 30 minutes, necessary to allow the correct interaction
between the antibody and its antigen, represented by the progesterone moieties anchored to
the gold substrate. The buffer was not degassed again, for the same reasons previously
examined312. After this exposure time, the chip was rinsed with sEBSS + 0% BSA, to remove any
unbound antibody still present in the sensor cell and the binding capacity was recorded. The
recorded sensorgram is reported in Figure 4.11.
CHAPTER 5 139
Figure 5.11 - SPR sensorgrams for the binding of anti-mouse progesterone antibody to the
progesterone-C7-4KC:EG6OH at 1:40 solution ratio, in sEBSS + 0% BSA, under OC (no applied
potential), ON (+0.3 V) and OFF (−0.4 V) conditions.
The binding capacity (BC) and the switching efficiency (SE %) in sEBSS + 0% BSA were
calculated using Equation 2, to obtain values of 1091±94 and 70±4%. The averages and the
standard errors in the reported binding capacity were calculated from at least three different
SPR measurements. The SPR data collected for the four different buffer conditions are reported
in Table 5.3, alongside the calculated switching efficiencies.
CHAPTER 5 140
Table 5.3 – SPR data and switching efficiency of the progesterone-C7-4KC:EG6OH mixed
SAMs in PBS, sEBSS + 0% BSA, sEBSS + 0.1% BSA and sEBSS + 0.3% BSA respectively
PBS
Binding Capacity Switching Efficiency
1195±22 73±3%
sEBSS + 0% BSA
Binding Capacity Switching Efficiency
1091±94 70±4%
sEBSS + 0.1% BSA
Binding Capacity Switching Efficiency
1291±78 44±8%
sEBSS + 0.3% BSA
Binding Capacity Switching Efficiency
1048±286 35±10%
A comparison of the switching behaviour presented by the 1:40 progesteron-C7-
4KC:EG6OH solution ratio in the different buffers tested is reported in Figure 5.12.
CHAPTER 5 141
Figure 5.12 – Bar chart reporting the switching efficiency of the progesterone-C7-4KC:EG6OH
mixed SAMs in PBS, sEBSS + 0% BSA, sEBSS + 0.1% BSA and sEBSS + 0.3% BSA respectively.
The results showed that the switching efficiency in sEBSS + 0% BSA is comparable to the
one obtained in PBS and confirmed our hypothesis stating the possible influence of a charged
protein such as BSA in the molecular rearrangement over the gold substrate when an electrical
potential is applied.
CHAPTER 5 142
4.5 Conclusions and Future Work
Immunosensors are a well-studied and reliable platform to control the interactions
between a protein (Ab) and a small molecule (Ag) and Surface Plasmon Resonance (SPR) is
widely used as immunosensing analytical technique297,349,365,366. In this work, the findings of our
previous research194,196–199 were applied to the aim of creating a switchable platform able to
control the exposure of an antigen molecule (progesterone) on the surface and the binding to
its antibody in solution (anti-mouse progesterone antibody), upon the application of an
electrical potential. It was successfully demonstrated that the 4-Lys oligopeptide molecule used
in our previous studies, is able to expose or conceal on demand the moieties of progesterone,
leading to a selective control over the antibody binding, upon the application of an electrical
potential, never demonstrated before. However, its switching activity is influenced by both the
presence of a spacer between the switching oligopeptide and the end group and the presence
of Bovine Serum Albumin (BSA) in the buffer.
Firstly, the spacer of 8-carbon atoms, increases the gap distance between the
progesterone end group and the ethylene glycol thiol (EG6OH) matrix. In addition, this alkyl
chain is not influenced by the application of an electrical potential to the gold substrate. When
no potential is applied (OC conditions), the alkyl spacer can lead to intercrossing on the gold
surface, between the progesterone-C7-4KC chains, leading to a lower binding capacity
compared to the one recorded for biotin-4KC in the same conditions. When an electrical
potential is applied, the alkyl chains of the spacer are neither attracted towards or repelled
from the surface, leading to an undesired orientation of the progesterone molecules and
resulting into a lower switching efficiency, compared to the one recorded for the same solution
ratios of biotin-4KC, in the same conditions (PBS buffer).
CHAPTER 5 143
Secondly, from our findings we can infer that the presence of BSA in the buffer used for
the switching studies, greatly influences the switching performances of the studied
progesterone-C7-4KC:EG6OH system. This phenomenon can be attributed to overall negative
charge presented by BSA at pH 7363, that could permit the interaction between this protein
and either the positively-charged oligolysine peptide chains and/or the negatively-charged
gold surface, when the -0.4 V potential is applied364. However, the complete removal of BSA
from the sEBSS buffer could influence the motility and the function of spermatozoa367,368 in the
future switching studies aimed to control the calcium ion influx and the hyperactivation in
sperm cells.
Starting from the results obtained in this chapter, our future work will be directed to
the understanding of: 1) the influence of the alkyl spacer length on the switching efficiency, by
testing the binding of anti-mouse progesterone antibody to different
progesterone-Cn-4KC:EG6OH mixed SAM (with n either < or > 8) by electrochemical SPR and 2)
the ability of different progesterone-Cn-4KC:EG6OH mixed SAM (with n either < or > 8) to trigger
the calcium ion influx and hyperactivation in sperm cells.
The results illustrated in this chapter will create the basis for the development of novel
immunosensing platforms, applicable to biomedicine, such as the studies of autoimmune
diseases and several pathologies involving the interaction between antigens and antibodies.
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Chapter 6 – Orthogonal Functionalisation of Surfaces for Controlling Sperm Cell
Adhesion and Hyperactivation
Abstract: This chapter describes the development of an orthogonal functionalisation method of
a glass-gold micropattern, for the control of both sperm cells adhesion and hyperactivation. The
cells adhesion was studied on two different surfaces, composed by poly-D-lysine (PDL) or silane-
PDL layers, to select the most suitable one for functionalisation purposes. Each step of the
orthogonal functionalisation was studied in detail and each surface was characterised by XPS.
This work shows the importance of an appropriate orthogonal molecule selection, to achieve
the desired surface properties. This study represents a starting point for developing effective
orthogonal surfaces able to control cellular response and activity.
6.1 Introduction
The continuous advancements in nanotechnology set new challenges in the
development of new surfaces with multiple bioactive molecules, intended for cell biology and
nanomedicine applications369. To create a bi- or multifunctional platform, different chemistries
are needed, especially if the platform is composed by different substrate materials. The use of
a multi-material substrate enables a better and efficient control over the localisation of the
different biomolecules. This control permits to create surfaces with well-defined and desired
density, spacing and molecular orientation370. Such surfaces can be tailored to present a large
variety of features making them flexible for a vast range of chemical and biological conditions
and purposes, ranging from biosensors to microarrays 369,371. Being some substrates negatively
affected by the reiteration of chemical reactions on the surface, it is necessary to reduce the
number of reaction steps, the use of protective groups and create simple functionalisation
protocols. Different techniques are used to achieve the orthogonal functionalisation of
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substrates, such as photolithography372, soft373 and electron-beam lithography374, dip-pen
nanolithography62 and micro-contact printing (CP)121,122,370. Several challenges must be
addressed in building such complex molecular architectures: the types of chemistry used could
interfere with each other or lead to undesired side-products, the conditions must be mild and
often biocompatible, the patterning techniques could damage the molecules present on the
surfaces (e.g. UV irradiation in photolithography370). Orthogonal self-assembly of molecules
was demonstrated for the first time in 1989 by Whitesides79 and it has now become a major
research focus especially in the preparation of devices able to enhance the signal in localised
SPR (LSPR). Orthogonal ligation chemistry permits the creation of pre-patterned surfaces with
multifunctional characteristics, exploitable to immobilise molecules on discrete areas, in a
specific manner, under mild conditions370.
Several examples of orthogonally functionalised surfaces have been reported in the
literature in the last decade375–377, but a noteworthy work for the aim of this research project,
was the one done by Durán and co-workers on polysilicon-gold chips378. They created a bi-
functional platform displaying two different biomolecules on the same device, with potential
employability as biosensor, biomarker or therapeutic agent378. By using a five-steps process,
the polysilicon area of the device was functionalised with molecules of
11-(triethoxysilyl)undecanal (TESUD) carrying a fluorescein conjugate concanavalin (F-ConA)
fluorescent tag, whereas on the gold area of the same device a SAM of mercaptoundecanoate-
NHS that was then linked to a Texas Red conjugated WGA (TR-WGA) molecule (Figure 6.1).
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Figure 6.1 – Steps involved in the preparation of double biofunctionalized chips. (a)
Polysilicon–chromium–gold chip, (b) Polysilicon surfaces activation by piranha cleaning, (c)
Mercaptoundecanoate-NHS SAM formation on gold, (d) TR-WGA immobilization via amide
bond formation, (e) TESUD SAM formation on polysilicon substrates and (f) F-ConA
immobilization via amide bond formation.
The successful completion of the orthogonal bi-functionalisation was then verified by
fluorescent microscopy. Herein, it is illustrated the preliminary studies performed to plan an
orthogonal functionalisation procedure of gold-glass microelectrodes. Our work is aiming to
create a molecular architecture containing both positively molecules of poly-D-lysine (PDL) for
sperm cells adhesion and a switchable mixed self-assembled monolayer that can be tuned to
control the sperm cells response to progesterone.
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6.2 Objectives
1) To study and optimise the adhesion of sperm cells on glass substrates, reproducing the
glass areas of a micropatterned surface.
2) To form the oligopeptide mixed SAM progesterone-C7-4KC:EG6OH previously studied
on gold substrates, reproducing the gold areas of a micropatterned surface.
3) To fully characterise the surfaces prepared in objectives 1 and 2 via XPS, contact angle
and ellipsometry.
4) To analyse the feasibility of the proposed orthogonal functionalisation strategy.
6.3 Results and Discussion
6.3.1 Study of sperm cells attachment on poly-D-lysine and silane/poly-D-lysine layers and
surface characterisation
Both poly-D-lysine (PDL) coating and silane-PDL layers were studied as an option for
sperm cells immobilisation, to analyse the effect on cell adhesion of the different bonding of
PDL to the surface. In the case of PDL coating, a layer of PDL is physisorbed on the glass
substrate, whereas in the case of silane-PDL, poly-lysine molecules are linked to the glass
surface thanks to strong covalent glass-silane and silane acid groups-lysine amino groups
bonds.
One of our future aims will be the use of the results obtained in this chapter to plan the
step for the orthogonal functionalisation of a micropatterned electrode. The orthogonal
functionalisation of a surface usually involves several steps329. Having the desired molecules
covalently bonded will avoid the risk of layer removal during the process.
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Cleaned glass microscope slides were functionalised with 1) a layer of poly-D-lysine
(PDL) coating (Figure 6.2), obtained upon evaporation of a PDL solution in UHQ water (0.1
mg/ml) at room temperature, presenting weak electrostatic interaction between charged
amine groups and the glass surfaces and 2) a layer of carboxyethylsilanetriol was first deposited
by chemical vapour deposition (CVD) to form strong covalent bonds between the hydroxyl
groups on glass and silicon atoms in the silane molecules. The carboxylic groups present on the
silane molecules were then bound to PDL molecules via carboxylic acid activation with
EDC/NHS, to form covalent amide bonds, as described in “Chapter 3 – Experimental Procedures
and Protocols”.
Figure 6.2 – Chemical structure of monomer units composing poly-D-lysine (PDL)
Poly-D-lysine is a polymer of molecular weight >300,000 Da, composed by lysine
aminoacid monomers. The lysine residues provide a positively charged backbone, due to the
presence of protonated amino groups (NH3+) at pH 7, below the amino pKa of 9.06, which can
interact with the negatively charged sites present on the sperm cells membrane, supporting
adhesion. It is widely used in cell research as standard method for facilitating cell
attachment379–381.
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6.3.1.1 XPS characterisation of poly-D-lysine and silane/poly-D-lysine layers
The surfaces used in the study of sperm cells attachment were analysed by XPS, that
revealed the presence of the elemental species C, N, O and Si on both layers, confirming the
formation of both PDL and silane-PDL layers. The C 1s spectrum (Figure 6.3 a) can be
deconvoluted into three peaks, corresponding to three different binding environments. The
peak at 284.8 eV is attributed to C-C bonds334,335, while the peak at 286.1 eV corresponds to C-
N binding environment. The third peak at 288.0 eV can be assigned to the C=O moiety, that
could be assigned to sodium carbonate, naturally present in glass substrates 334,335,382. The N 1s
spectrum (Figure 6.3 b) consist in a large peak that can be deconvoluted into three peaks. Due
to high concentration of amino groups on the surface, the nitrogen peak is intense, and three
peaks are observable. The first peak is centred at 401.0 eV, can be assigned to protonated
amino groups (NH3+), whereas the second peak at 399.7 eV corresponds to amide (CONH) and
amino (NH2) groups383, the third peak at 397.7 eV is ascribable to silicon nitrates that can be a
component of glass312. The O 1s spectrum (Figure 6.3 c) can be deconvoluted into two peaks,
assignable to C-O groups (531.6 eV) and C=O plus SiOx (533.0 eV), due to silicon oxides being
naturally present in glass substrates334,335. The peak at 535 eV can be assigned to the sodium
Auger peak (Na KLL), that could be assigned to sodium carbonate, one of the components of
glass.334,335,382. In this case, the Si 2p spectrum (Figure 6.3 d) is only attributable to the silicon
oxides SiOx (103.0 eV) and SiOH (103.8 eV) groups naturally present in the glass
substrate116,334,335,383–391. The small shift of the N, O and Si peaks (less than 0.5 eV) is due to the
limited conductivity of glass, causing a charge accumulation on the surface392.
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Figure 6.3 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions of pure 0.5 mg/ml
PDL layers on glass substrates.
The XPS analysis was also performed on silane-PDL layers (Figure 6.4) on glass
substrates, revealing the presence of the elemental species C, Si, N and O, confirming the
formation of the layer.
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Figure 6.4 – Schematic representation of silane-PDL layers on glass substrates
The C 1s spectrum (Figure 6.5 a) is formed by three peaks, which are attributable to the
following binding environments334,335: C-C and C-Si (284.8 eV)393, C-N (286.4 eV) and C=O (288.3
eV). The Si 2p spectrum (Figure 6.5 b) is formed by two peaks, that can be assigned to the
binding environments of SiOx and Si-O-Si groups (103.4 eV) and Si-C and Si-OH (102.5
eV)116,334,335,383–391. The N 1s spectrum (Figure 6.5 c) can be deconvoluted into two peaks, which
support the presence of lysine moieties on the surface. The first peak at 399.0 eV can be
assigned to amino (NH2) and amide (CONH) groups. The second peak at 401.6 eV corresponds
to protonated amino groups (NH3+)383. The silicon nitrates peak coming from the glass itself
cannot be distinguished from the background noise. This difference can be due to the presence
of silane-PDL molecules covering the glass surface. It can in fact be noticed the higher intensity
of the protonated amino groups (NH3+) peak, indicating a high presence of PDL on the surface.
The O 1s spectrum (Figure 6.5 d) can be deconvoluted into two different peaks, corresponding
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to three binding environments. The first peak centred at 532.7 eV can be assigned to Si-O and
C=O bonds and the second peak at 531.5 eV can be assigned to Si-OH moieties334,335. The
limited conductivity associated with the glass substrate, cause a charge accumulation on the
surface that leads to small binding shifts (less than 0.5 eV), if compared to the peak values
obtained on gold substrates392.
Figure 6.5 – XPS spectra of the a) C 1s, b) Si 2p, c) N 1s and d) O 1s regions of silane-PDL layers
on glass substrates.
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6.3.1.2 Contact angle and ellipsometry analysis of poly-D-lysine and silane/poly-D-
lysine layers
The formation of poly-D-lysine (PDL) and silane-PDL layers were analysed by contact
angle and ellipsometry. As expected, the water advancing (Adv) and receding (Rec) contact
angles for silane-PDL layers revealed hydrophilic characteristics, whereas it was impossible to
measure the contact angle for the layer of pure PDL, since the water droplet re-dissolved the
PDL itself (Table 6.1).
Ellipsometry analysis confirmed the formation of both PDL and silane-PDL layers, but,
as expected, the homogeneity of both layers on surface was poor, due to the lack of control
over the molecular organisation on surfaces. The formation of pure silane layer resulted in an
ellipsometric thickness of 1.7 ± 0.2 nm which is significantly higher that the theoretical
molecular length of 0.53 nm. This difference can be explained with the formation of a possible
tri-layer of silane, instead of a monolayer. The surfaces were formed on silicon wafer
substrates, to allow the ellipsometry measurements. Ellipsometry measurements cannot be
performed on glass, due to the transparency of the surface. Silicon wafers are not transparent
and form covalent bonds with silane molecules, in the same way glass surfaces do. The exact
theoretical molecular length of poly-D-lysine cannot be calculated, because the number of
lysine units in the polymer is not exactly known
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Table 6.1 - Advancing and receding water contact angle and ellipsometric thickness for the
different layers formed for 24h. The theoretical molecular lengths were calculated using
ChemBio3D Ultra 12.0 in which the molecules were in fully extended conformation. The
averages and standard errors were calculated from at least three different measurements.
Layer Contact Angle Thickness (nm)
Adv. Rec. Theor. Measured
Silane 61° ± 2 56° ± 3 0.53 1.7 ± 0.2
Silane-PDL 70° ± 5 63° ± 3 - 5.8 ± 2
PDL - - - 16 ± 4
The contact angle results indicate that both silane and silane-PDL layer show slightly
hydrophilic properties as expected. The large hysteresis (θAdv – θRec) of 5˚ for the silane layer
and 7˚ for the silane-PDL layer are an indication of the low order of both layers. In addition,
when PDL is bond to the surface, the contact angle become more hydrophobic and measured
thickness increases, confirming a change in the layer, that can indicate the successful
attachment of PDL.
The measured thickness of the silane layer is higher than the theoretical thickness
calculated using ChemDraw 3D. It is possible to state that the resulting silane layer is not a
monolayer, result that can be attributed to the lack of control over the chemical vapour
deposition conditions.
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6.3.1.4 Effect of different solvents on poly-D-lysine and silane/poly-D-lysine layers
and sperm cells attachment
Sperm cells were prepared following the procedure described in detail in Chapter 6.
300 l of fresh sperm sample where first added to 700 l of EBSS buffer containing 0.3% BSA
in several falcon tubes and left in the incubator for an hour, to allow sperm cells to lose the
cholesterol layer on cell membrane. After this first step, the top layer (swim-up) containing
sperm cells, was collected from each falcon tube and left in the incubator to capacitate for two
hours. Then 300 l of cell suspension, containing capacitated sperm cells labelled with Calcium
Green 1-AM, were deposited on glass microscope slides functionalised with 1) Poly-D-lysine
coating and 2) silane/Poly-D-lysine layers with PDL concentrations of 0.1 mg/ml and 0.5 mg/ml
to compare cell adhesion (section 3.3.1). The microscope slides were attached with vacuum
grease to a purpose-built, perfusable, polycarbonate imaging chamber (section 3.3.3). The
imaging chamber is equipped with two openings, one for the buffer entering the chamber and
one for the buffer leaving the chamber, sucked away by a vacuum pump. It also contains a well
to allocate the reference electrode and an opening to allocate the platinum counter electrode.
The imaging chamber containing sperm cells was left in a wet chamber at room temperature
for 30 minutes before being mounted on the microscope, connected to the perfusion system.
After the incubation time, the surface was rinsed with sEBSS for 10 minutes at a flow rate of
approximately 1 ml/min for at least 15 min, to remove loose sperm cells present on the surface.
A roller pump push sEBSS solution into the chamber and overflow is removed by a suction
pump with trap from the exit port (Figure 6.6).
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Figure 6.6 – Schematic representation of the imaging chamber mounted on the microscope
slide and connected to the perfusion system.
The sperm cells attached on the two surfaces tested were then analysed by
fluorescence microscopy. Snapshots of different areas of the surface are collected and cell
counted on each field. From the images of the two surfaces collected, it is possible to assert
that Poly-D-lysine coating is more effective in fostering cell adhesion when compared to
silane/PDL layers (Figure 6.7).
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Figure 6.7 – Fluorescence images of cell adhered on PDL coated-surfaces (left), silane-PDL
layers formed using a PDL concentration of 0.1 mg/ml (centre) and silane-PDL layers formed
using a PDL concentration of 0.5 mg/ml (right).
The average number of cells per field in the case of the PDL coating, is 83±7, whereas in
the case of silane-PDL (0.1 mg/ml PDL) and silane-PDL (0.5 mg/ml) is 9±4 and 34±5 respectively.
The average and the error were calculated based on three different fields of the same sperm
sample. The results indicate that the PDL coating is more effective in promoting cell adhesion,
whereas on the silane-PDL layer prepared with a PDL concentration of 0.1 mg/ml cell adhesion
is scarce. This result is likely due to the presence of a smaller number of charged amino groups
free on the surface for interaction with sperm cells membrane.
To understand if the ethanol used as solvent in the formation of Progesterone-C7-
4KC:EG6OH mixed SAM, can influence cell adhesion, glass microscope slides treated with both
PDL and silane-PDL (0.5 mg/ml of PDL) were immersed in HPLC ethanol overnight. Both surfaces
were then tested for cell adhesion, following the procedure described above. By observing the
two surfaces, it is possible to notice that ethanol has not an important effect on cell attachment
on silane-PDL layers, whereas it seems to affect the quality of the PDL coating. “Stripes” are
clearly visible on the surface coated with 0.1 mg/ml of PDL. In addition, the sperm cells look
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poorly labelled and less active if compared to the previous experiment, before the ethanol
rinsing of the surface. The worse labelling and the poorer activity can also be due to cell starting
naturally to die after being kept in the incubator for a long time (Figure 5.8).
Figure 6.8 – Fluorescence images of cell adhered on PDL coated-surfaces rinsed with ethanol
(left), and silane-PDL layers formed using a PDL concentration of 0.5 mg/ml rinsed with
ethanol (right).
From the picture of the surface, it can be inferred that the presence of ethanol can
affect the quality of the PDL coating. The coating appears to be partially re-dissolved by the
small percentage of water present in the solvent, or by the ethanol itself, being poly-lysines
slightly soluble in ethanol394. The silane-PDL surface (0.5 mg/ml of PDL) does not present the
same kind of film effect after the ethanol rinsing. In addition, cells appear active and well
labelled. The number of cells attached on the surfaces coated with PDL is still greater if
compared to that on silane-PDL (0.5 mg/ml) surfaces. However, it is reasonable to hypothesise
that the visible change of morphology of the non-covalently formed PDL film on glass, could
then affect the switchable moieties on the gold areas of the micropattern. The silane-PDL layer
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was therefore chosen to functionalise the glass areas of the micropattern, to promote cell
adhesion.
6.3.2 XPS characterisation of clean glass and gold substrates
Glass and gold substrates were chosen as model systems to study the fabrication
procedure, due to the impossibility of performing XPS analysis on the micropatterned surfaces.
XPS characterisation of glass and gold substrates cleaned in piranha solution was performed to
be used as references.
Clean glass
XPS analysis of clean glass substrates showed the presence of elemental species C, N,
O, S and Si. The C, N, O and Si peaks are assignable both to substances naturally present in
glass, such as carbonates and silanes and to molecules contaminating the laboratory
environment334,335,382. The C 1s spectrum (Figure 6.9 a) can be deconvoluted into three peaks,
assignable to C-C and C-Si (284.8 eV), C-O (286.7 eV) and C=O (288.2 eV) groups334,335. The N 1s
spectrum (Figure 6.9 b) consist in a peak centred at 400.5 eV that can be assigned to nitrogen
coming from the air (N2)383. The O 1s spectrum (Figure 6.9 c) consists in a peak centred at 532.9
eV that can be assigned to silicon oxides (SiOx) present in the glass and to organic C=O
moieties334,335. The C, N and O signals can be due to contamination coming either from the air
or the laboratory environment. The S 2s spectrum (Figure 6.9 d) consists of a peak centred at
233.4 eV334,335,395, indicating an unexpected presence of sulphur on the surface. The S 2p signal
was not recorded, since it is shielded by the Si 2s intense signal. The sulphur can either be due
to contamination coming from the laboratory environment or piranha solution residues
remained on the glass substrate after the rinsing. The Si 2p spectrum (Figure 6.9 e) can be
deconvoluted into two peaks, the first one centred at 103.2 eV, can be assigned to silicon
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hydroxides (SiOH) and the second one centred at 103.8 eV can be assigned to silicon oxides
SiOx and Si-O-Si groups, both present in the glass substrate116,334,335,383–391.
Figure 6.9 - XPS spectra of the a) C 1s, b) N 1s, c) O, d) S 2s and e) Si 2p regions recorded on
clean glass substrates.
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The ratios between the elements analysed (N/C, O/C, S/C and Si/C) were calculated to
obtain reference ratios to be used as a comparison in the next steps. The calculated values are
0.13±0.03 for N/C, 33.42±2.23 for O/C, 0.18±0.03 for S/C and 5.76±0.13 for Si/C respectively.
The results show a large presence of oxygen and silicon on clean glass substrates, due
to the glass composition, as expected334,335,382. There is also a small amount of sulphur,
attributable to either contamination from thiols in the laboratory atmosphere or to piranha
solution residues. Finally, noticeable is the much larger amount of oxygen than the carbon
amount, attributable to both the silicon oxides and carbonates naturally composing glass
substrates334,335,382.
Clean gold
XPS analysis of clean gold substrates showed the presence of elemental species C, N, O
and S. The C, O and peaks can be the result of contamination due to handling and/or the
presence volatile molecules present in the laboratory environment. The C 1s spectrum (Figure
6.10 a) can be deconvoluted into two peaks, assignable to C-C (284.4 eV) and C-O (286.2 eV)
groups334,335. The N 1s spectrum (Figure 6.10 b) consist in a small peak centred at 399.8 eV that
can be assigned to both nitrogen gas and nitrogen-containing volatile molecules present in the
air of the laboratory383. The O 1s spectrum (Figure 6.10 c) consists in a small peak centred at
532.0 eV that can be assigned to organic C-O moieties334,335. The S 2p spectrum (Figure 6.10 d)
consists of a doublet peak centred at 162.1 and 163.3 eV, indicating an unexpected presence
of unbound sulphur on the surface395. The sulphur can either be due to contamination coming
from volatile thiols present in the laboratory atmosphere. No presence of silicon was recorded.
CHAPTER 6 162
Figure 6.10 - XPS spectra of the a) C 1s, b) N 1s, c) O and d) S 2p regions recorded on clean
gold substrates.
The ratios between carbon and the other elements (N, O and S) were calculated to
obtain reference ratios to be used as a comparison in the next steps.
The calculated N/C and S/C ratios of 0.05±0.02 and 0.08±0.01, respectively, show a small
presence of both nitrogen and sulphur if compared to the amount of carbon. In addition, the
O/C ratio of 0.20±0.10 confirms a high presence of carbon if compared to the other elements.
The recorded carbon can either be due to organic contamination from the laboratory of from
handling of the samples. The foil used to wrap the samples for shipment, could have transferred
some organic compounds to the surfaces.
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6.3.3 XPS characterisation of each step performed on either plain gold or glass surfaces
The formation of the two different surfaces on the glass and gold was done via the
following steps:
1) Deposition of a 11-mercapto-1-undecanol protective SAM on gold.
2) Formation of a silane-PDL layer on glass.
3) Removal of the protective SAM by applying a potential of -1.5V for ten minutes to the
gold microelectrodes.
4) Formation of progesterone-C7-4KC:EG6OH mixed SAM on gold.
Both gold and glass were exposed to all these steps to simulate what will happen on the
micropatterned substrates. To understand if each step can affect either the sperm cells
adhesion and the achievement of the final molecular structure on the micropattern, a detailed
XPS analysis was made.
6.3.3.1 First step: protective thiol on gold and glass
Gold substrates:
The XPS analysis was performed on 11-mercapto-1-undecanol SAMs on gold, revealing
the presence of the elemental species S, C and O, confirming the presence of thiol molecules
on the surface. The S 2p spectrum (Figure 6.11 a) consists of two doublet peaks, with the first
one at 163.2 eV (S 2p1/2) and 162.0 eV (S 2p3/2) assignable to sulphur chemisorbed on the gold
surface (S-Au)395,396. The second doublet peak centred at 164.3 eV (S 2p1/2) and 163.1 eV (S
2p3/2) can be ascribed to unbound sulphur on the gold surface (S-H)395,396. The C 1s spectrum
(Figure 6.11 b) is formed by two peaks, which are attributable to the following binding
environments: C-C (284.7 eV) and C-S, C-N and C-O (286.4 eV)334,335. The O 1s spectrum (Figure
6.11 c) is formed by a peak centred at 532.7 eV corresponding to C-O334,335. No presence of N
CHAPTER 6 164
was recorded, as expected (Figure 6.11 d). The difference in the nitrogen presence compared
to the clean gold case can be explained with the ease for clean gold to absorb volatile molecules
from the air. On the contrary, the presence of a well-packed matrix of alkyl thiol prevents this
contamination phenomenon. These peaks will be used as reference to analyse the changes on
the substrates, as a result of the formation of the different layers on the gold substrates.
Figure 6.11 – XPS spectra of the a) S 2p, b) C 1s, c) O 1s and d) N 1s regions of
11-mercapto-1-undecanol SAMs on gold.
The calculated O/C and S/C ratios were compared with the ones calculated from the analysis
of clean gold to verify if the 11-mercapto-1-undecal monolayer was formed correctly.
CHAPTER 6 165
Every molecule of 11-mercapto-1-undecanol (Figure 6.12) contains 11 C atoms, 1 O
atom and 1 S atom, from which it is possible to calculate the both the expected C/O and C/S as
equal to 11.
Figure 6.12 – Molecular structure of 11-mercapto-1-undecanol
The calculated O/C ratio of 0.28±0.01 shows no significant change if compared to the
one calculated for the clean gold (0.20±0.10). In addition, both the calculated C/O and C/S
ratios (3.55±0.09 and 9.25±0.90) are smaller than the expected C/O and C/S ratios for 11-
mercapto-1-undecanol (both equal to 11), showing that the desired MUD monolayer did not
form correctly on the gold substrates. It is reasonable to infer that there was an additional
contribution to the carbon content due to contamination.
Glass substrates:
The XPS analysis of 11-mercapto-1-undecanol SAMs on glass, confirmed that no
monolayer was formed as S 2s peaks were not present. However, the XPS analysis of clean glass
substrate showed the presence of sulphur on the surface. This discrepancy can be due to the
different time of preparation and the presence of thiol contaminants in the atmosphere of the
laboratory. The C, N, O and Si peaks recorded are ascribable both to substances naturally
present in glass, such as carbonates and silanes and to the nitrogen present in the air (Figure
5.9)334,335,382. The C 1s spectrum (Figure 6.13 a) is formed by three peaks, which are attributable
to the following binding environments: C-C and C-Si (284.6 eV), C-O (286.5 eV) and C=O (288.1
CHAPTER 6 166
eV)334,335. The Si 2p spectrum (Figure 6.13 b) can be deconvoluted into two peaks, attributed
to silicon oxides (SiOx) and Si-O-Si groups (103.3 eV) and Si-OH groups (102.7 eV), present on
the glass substrates116,383,385–387,389–391. The N 1s spectrum (Figure 6.13 c) is characterised by a
high background noise and a single peak centred at 400.2 eV can be identified. This peak can
be assigned to the nitrogen in the air. The S 2s spectrum (Figure 6.13 d) indicates that sulphur
is not present on the surface. The O 1s spectrum (Figure 6.13 e) is formed by a peak centred at
532.7 eV corresponding to C-O334,335.The S 2p spectrum could not be recorded since it is
shielded by the intense Si 2s peak. However, the absence of the S 2s peak already suggested
the absence of sulphur residues on the surface. These peaks will be used as reference to analyse
the changes on the substrates, as a result of the formation of the different layers on the glass
substrates.
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Figure 6.13 – XPS spectra of the a) C 1s, b) Si 2p, c) N 1s, d) S 2s and e) O 1s regions of
11-mercapto-1-undecanol SAMs on glass.
The ratios between the elements analysed (C, N, O and Si) were calculated and
compared to the ones obtained from the analysis of clean glass substrates.
CHAPTER 6 168
The results show an increase in the carbon amount on glass surfaces after the protective
thiol immobilisation, compared to the carbon present on clean glass surfaces. In fact, the C/N
ratios went from 7.8±1.9 to 24.4±3.11. There is also a significant decrease in the oxygen
amount, since the O/C ratio decreased from 33.42±2.23 to 0.12±0.01. It is possible that some
molecules of 11-mercapto-1-undecanol are physisorbed on the glass substrates and the rinsing
with ethanol is not enough to remove them. However, the signal of S 2p cannot be recorded
due to the strong Si 2s shielding and the S 2s signal is too noisy to identify the presence of
sulphur on the glass surface.
6.3.3.2 Second step: silane-PDL on gold and glass
The silane-PDL layer was formed following the procedure illustrated in Chapter 3 –
Experimental Procedures and Protocols. The silane molecules were deposited on the substrates
via Chemical Vapour Deposition (CVD) by incubating either the gold or glass substrates in a
vacuum chamber in silane atmosphere for an hour. After the deposition step, the silane layer
was cured by leaving the functionalised substrates in the vacuum oven at 100˚C for 30 minutes.
The substrates were then immersed in a 1mM HCl solution in UHQ water for 5’ under
gentle shaking, to form carboxylic acid groups on the surface and then immersed in a solution
1:1 of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide (EDC/NHS)
overnight as illustrated in section 6.3.1.3.
Gold substrates:
The XPS analysis was performed on silane-PDL layer formed after the deposition of 11-
mercapto-1-undecanol SAMs on gold, revealing the presence of the elemental species C, N, O,
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S and Si. The C 1s spectrum is formed by three peaks (Figure 6.14 a), which are attributable to
the following binding environments334,335: C-C and C-Si (284.8 eV) and C-S, C-N and C-O (286.3
eV), the third small peak at 288 eV, corresponds to C=O groups and it can be attributed to the
carboxyl group on the silane or the carboxyl groups on the PDL chain334,335,397. The N 1s
spectrum (Figure 6.14 b) consists in one small peak centred at 400.2 eV383, that can be
attributed either to the presence of poly-D-lysine on the gold surface or to the nitrogen present
in the air.383. The O 1s spectrum (Figure 6.14 c) is formed by two peaks334,335, the peak centred
at 532.4 eV can be assigned to both C-O and Si-OH groups and the small peak at 533.0 eV is
ascribable to both C=O and Si-O-Si groups, indicating the presence of either silane or silane-
PDL on the surface. The S 2p spectrum (Figure 6.14 d) consists of a doublet peak at 163.4 eV (S
2p1/2) and 162.3 eV (S 2p3/2), indicating that there a small quantity of sulphur chemisorbed on
the gold surface, corresponding to the presence of the thiol on the surface395. However, the
peak at 163.4 eV can also be assigned to unbound sulphur on the gold surface, but the relative
doublet peak could not be fitted using CASA XPS, due to the shielding effect of the Si 2s peak.
The Si 2s spectrum (Figure 6.14 e) is formed by a peak centred at 153.5 eV showing the possible
presence of unexpected silane on the gold surface 334,335,384. The presence of the silicon peak
can be due to the unwanted deposition of silane on the whole or part of the gold substrate.
The Si 2p could not be recorded, since its regions overlaps with the Au 5d region116,334,335,383–
391. It must be highlighted that in this case the Si 2s and S 2p signal were recorded together in
the same region, leading to a high background noise in the less intense sulphur peak, compared
to the silicon one. The recorded results suggest that the second step of the fabrication, that
should result in the formation of a silane layer only on the glass substrate, also affect the gold
substrate. This could lead to the presence of PDL on the gold surface, resulting in the cell
attachment on both glass and gold substrates.
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Figure 6.14 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s, d) S 2p and e) Si 2s regions of silane-
PDL SAMs on gold after the first step.
The calculated ratios determined after this step were compared to the ones calculated
after the deposition of the protective thiol, to analyse if the changes on the glass surface could
be ascribable to the effective formation of the desired silane-PDL layer.
CHAPTER 6 171
The O/C ratio shows an increment of the oxygen presence on the gold substrate that
went from 0.28±0.01 to 0.62±0.05, together with a small decrease of the sulphur amount, since
the S/C ratio moved from 0.11±0.01 to 0.08±0.01. Each molecule of silane contains four oxygen
atoms and each amide bond on lysine moieties coming from PDL contains an oxygen atom. The
O/C ratio increases from 0.28±0.01 to 0.62±0.05 and nitrogen is present, even if in small
quantities, whereas before its peak cannot be recorded. This circumstance could be explained
by the physisorption of silane-PDL layer on the gold surface, causing also the shielding of the
sulphur peak by the Si 2s signal.
Glass substrates:
The XPS analysis was performed on silane-PDL layer formed after the deposition of 11-
mercapto-1-undecanol SAMs on glass, revealing the presence of the elemental species C, N, O
and Si, indicating the presence of the silane-PDL structure. The C 1s spectrum is formed by 3
peaks (Figure 6.15 a), corresponding to four binding environments334,335. The peak centred at
284.8 eV can be assigned to C-C and C-Si bonds397, the peak at 286.4 eV is ascribable to C-N
bonds in C-N groups, whereas the third peak at 288.3 eV is assigned to the C 1s photoelectron
of the carbonyl moiety, C=O. The N 1s spectrum (Figure 6.15 b) can be deconvoluted into two
peaks, the first one corresponding to amino and amide groups (400.0 eV) and the second one
ascribable to protonated amino groups (401.6 eV)383. The O 1s (Figure 6.15 c) composed by two
peaks, corresponding to three different binding environments334,335. The first peak at 531.2 eV
corresponds to Si-OH moieties, whereas the second peak centred at 532.7 eV can be assigned
to C=O and Si-O-Si groups. The Si 2p spectrum (Figure 6.15 d) can be deconvoluted into two
peaks, the first one centred at 102.6 eV can be ascribed to Si-OH groups, whereas the second
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peak centred at 103.3 can be assigned to SiOx present in the glass and Si-O-Si groups present
in the silane chains116,334,335,383–391. Again, the small binding shift (less than 0.5 eV) is caused by
the charge accumulation onto the glass substrate392.
Figure 6.15 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions of silane-PDL
SAMs on glass after the first step.
The ratios determined after this step were compared to the ratios calculated after the
deposition of the protective thiol, to analyse if the changes on the glass surface are ascribable
to the effective formation of the desired silane-PDL layer.
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The results show an increment of the amount of both nitrogen and oxygen on the glass
surface. In fact, the N/C ratio went from 0.040±0.004 to 0.12±0.02 and the O/C ratio increased
from 0.120±0.006 to 8.94±0.60. This is in accordance with the formation of a silane-PDL. In
addition, it is important to notice, taking into consideration the standard errors, that the Si/C
ratio (1.60±0.20) did not vary from the previous step (1.53±0.32). This phenomenon can be
explained by the increment of carbon amount to the same extent as silicon amount. However,
it is impossible to determine the quantity of silicon due to silicates present composing the glass
substrate itself. The presence of silane-PDL structures was also verified by studying the
attachment of sperm cells on glass surfaces (see section 6.3.4)
6.3.3.3 Third step: protective thiol removal
After the deposition of the silane-PDL layer, the 11-mercapto-1-undecanol thiol was
removed from the gold substrate, to prepare the surface for the functionalisation with
progesterone-C7-4KC:EG6OH mixed SAMs. The protective thiol was removed by applying a
potential of -1.5V for 10 minutes. Three different times of potential application were tested on
different gold substrates functionalised with 11-mercapto-1-undecanol SAM by 5 min, 10 min
and 20 min chronoamperometry and a cycle of cyclic voltammetry to verify the success of the
removal by the disappearance of the thiol desorption peak. The CV recorded on gold substrates
after the overnight incubation of the protective thiol, shows a clear reductive desorption peak
at -1.05 (black line on Figure 6.16). When the CV is recorded again, after the application of -
1.5V for 5, 10 and 20 min, the desorption peak has disappeared, indicating that the thiol has
been successfully removed from the gold surface. The 10-min time was chosen to both ensure
the complete removal of the thiol and avoid the damage of the gold substrate itself. However,
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cyclic voltammetry only shows the curve for thiol reduction and it is impossible to know if the
silane-PDL layer is still present on gold. The analysis of this step by XPS is therefore important
to study the effectiveness of the protective thiol removal step.
Figure 6.16 - Cyclic voltammetry of bare gold after piranha cleaning (orange line), after MUD
incubation (black line) and after 5, 10 and 20 minutes of -1.5 V chronoamperometry (green,
light blue and purple lines)
Due to the non-conductivity of glass substrates, after the second step the surfaces were
immersed for ten minutes in KOH 0.1M in water, to analyse if the basic solution could have any
effect on the silane-PDL layer. Both gold and glass surfaces were analysed by XPS.
Gold substrates:
The XPS analysis was performed on the gold substrates after the removal of the
protective thiol performed by applying a negative potential of -1.5 V for ten minutes in KOH
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0.1M in water. The results of the analysis revealed the presence of the elemental species S, C,
N, O and Si, indicating that the removal of the protective thiol and possibly some silane
residues, was not 100% successful as expected from CV results. The C 1s spectrum (Figure 6.17
a) can be deconvoluted into three peaks, corresponding to the following binding
environments334,335: the first peak centred at 284.7 eV can be ascribed to C-C and C-Si
moieties397, the second peak centred at 286.2 eV corresponds to C-N groups, whereas the small
peak at 288 eV can be assigned to the carbonyl moiety C=O, meaning that the electrochemical
treatment of the gold substrate did not completely remove the silane molecules. The N 1s
spectrum (Figure 6.17 b) consist in a small single peak centred at 400 eV, corresponding to
amino and amide groups383, indicating the presence of nitrogen on the surface, possibly to the
undesired presence of lysine molecules on the gold surface. The O 1s signal (Figure 6.17 c) is
represented by a small and large single peak centred at 532.3 eV that could be assigned to C=O
and Si-O-Si groups334,335. The Si 2s signal (Figure 6.17 d) consists of a single peak centred at
153.2 eV, indicating the undesired presence of silane on the surface. The Si 2p signal cannot be
recorded because is shielded by the Au 5d peak. The S 2p spectrum (Figure 6.17 e) has not a
high resolution, due to the shielding effect of Si 2s peak, but it can be deconvoluted into two
small peaks centred at 163.4 eV and 162.2 eV respectively. The first peak at 162.2 eV can be
assigned to a small amount of sulphur chemisorbed to the gold surface, whereas the second
one at 163.4 eV can be assigned to both bound sulphur and unbound sulphur. The doublet peak
of unbound sulphur could not be fitted using CASA XPS due to the shielding effect of Si 2s, but
it is reasonable to hypothesise the presence of free thiol groups on the gold substrate.
However, the signals of the elements C, N, O and S are recorded also on clean gold surface. This
make impossible to confirm the presence of protective thiol on the gold surface with certainty.
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On the contrary, silane is still present after the thiol removal, since no silicon signals were
recorded on pristine gold.
Figure 6.17 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s, d) Si 2s and e) S 2p regions after thiol
removal on gold substrates.
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The ratios between the elements analysed were calculated and compared to the ones
obtained from the previous step.
Both the calculated S/C and Si/C ratios, if the standard errors are taken into
consideration, indicate that no changes happened on the surface compared to the previous
step, in fact the S/C value is still 0.08±0.01 and the Si/C moved from 0.15±0.02 to 0.16±0.03. It
is possible to state that the removal of the protective thiol was unsuccessful and silane is still
present on the gold surface. This phenomenon could be due to the silane-PDL layer physisorbed
on the gold surface, preventing the correct removal of 11-mercapto-1-undecanol protective
thiol by cyclic voltammetry.
Glass substrates:
The XPS analysis was performed on the glass substrates after incubation, of the surfaces
obtained after the second step, in KOH 0.1M in water for ten minutes. The results of the
analysis revealed the presence of the elemental species C, N, O and Si. The C 1s spectrum
(Figure 6.18 a) can be deconvoluted into three peaks, assignable to different binding
environments334,335. The first peak centred at 284.8 eV corresponds to C-C and C-Si bonds, the
second peak centred at 286.4 eV is ascribable to C-N groups, whereas the third peak at 288.3
eV is assigned to the C 1s photoelectron of the carbonyl moiety, C=O. The N 1s spectrum (Figure
6.18 b) can be deconvoluted into two peaks, the first one corresponding to amino and amide
groups (400.0 eV) and the second one ascribable to protonated amino groups (401.2 eV)383.
The O 1s spectrum (Figure 6.18 c) is composed by two peaks, corresponding to three different
binding environments334,335. The first peak at 531.4 eV corresponds to Si-OH moieties, whereas
the second peak centred at 532.6 eV can be assigned to C=O and Si-O-Si groups. The Si 2p
spectrum (Figure 6.18 d) can be deconvoluted into two peaks, the first one centred at 102.6 eV
CHAPTER 6 178
can be ascribed to Si-OH groups, whereas the second peak centred at 103.4 eV can be assigned
to SiOx present in the glass and Si-O-Si groups present in the silane chains116,334,335,383–391.
Figure 6.18 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions incubation of
glass substrates in KOH for ten minutes, demonstrating that removal step will not affect the
integrity of the silane-PDL layer on the glass substrate.
The ratios between the elements analysed (C, N, O and Si) were calculated and
compared to the ones obtained from the analysis of the previous step on glass.
The results for the silicon element, demonstrate that the KOH solution does not affect
the silane-PDL layer on the glass substrate. The small change recorded in the Si/C ratio (from
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1.53±0.32 to 1.0±0.2) could be ascribable to a different surface coverage than the previous step
or a slightly different glass substrate composition. The calculated Si/C ratio of 1.0±0.2 shows
that there is as much carbon as silicon on the surface. However, it is again impossible to deduct
the quantity of both atoms arising from pure glass composition.
6.3.3.4 Fourth step: Progesterone-C7-4KC mixed SAM
Gold substrates
The XPS analysis was performed on the gold substrates incubated in Progesterone-C7-
4KC:EG6OH mixed SAMs (1:40 solution ratio), after the removal of the 11-mercapto-1-
undecanol SAM. The results of the analysis revealed the presence of the elemental species C,
N, O and S. The C 1s spectrum (Figure 6.19 a) can be deconvoluted into three peaks, assignable
to different binding environments334,335. The first peak centred at 284.6 eV corresponds to C-C
bonds, the second peak centred at 286.4 eV is ascribable to C-S, C-N and C-O groups, whereas
the third peak at 288.4 eV is assigned to the C 1s photoelectron of the carbonyl moiety, C=O.
The N 1s spectrum (Figure 6.19 b) consists in one small corresponding to amino (NH2) and
amide (CONH) groups (400.3 eV)383. The O 1s spectrum (Figure 6.19 c) is deconvoluted two
peaks, corresponding to two different binding environments334,335. The first peak at 532.8 eV
corresponds to C-O moieties, whereas the second peak centred at 531.8 eV can be assigned to
C=O groups. The Si 2p spectrum (Figure 6.19 d) can be deconvoluted into two peaks, the first
one centred at 102.6 eV can be ascribed to Si-OH groups, whereas the second peak centred at
103.4 eV can be assigned to SiOx present in the glass and Si-O-Si groups present in the silane
chains116,334,335,383–391. The presence of the silane peaks indicate that the silane-PDL layer was
not displaced by the presence of progesterone-C7-4KC and EG6OH thiol molecules. Finally, the
S 2s spectrum (Figure 6.19 e) was recorded instead of the S 2p spectrum, shielded by the
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intense Si 2s peak. The S 2s peak centred at 223.2 eV indicate the presence of sulphur on the
surface, but it does not discriminate between the sulphur covalently attached to the gold
substrate and the unbound sulphur.
CHAPTER 6 181
Figure 6.19 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s, d) Si 2p and e) S 2s regions after the
formation of Progesterone-C7-4KC:EG6OH mixed SAMs on gold substrates.
The calculated ratios after performing the last step on gold showed that there is not a
significant change in both C/N and C/Si ratios after this step. In fact, the C/N went from
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11.20±0.92 to 11.10±0.30 and the C/Si ratio went from 6.30±1.10 to 6.90±0.40. This can be due
to the presence of silane-PDL molecules on the gold substrate, that cannot be displaced by the
thiols present in the mixed SAM solution. The only element that increased is oxygen, in fact
the O/C ratio increased from 0.55±0.09 to 0.93±0.10. The theoretical O/C ratio for 1:40
progesterone-C7-4KC:EG6OH solution ratio is 0.21, that is lower than the one calculated from
the XPS experiments. The theoretical S/C ratio for the same mixed SAM solution ratio is 0.03,
that is in line with the calculated one of 0.04±0.03. However, the calculated standard error is
too high to assume that the desired mixed SAM has formed correctly on the gold substrates. In
fact, it is possible that the progesterone-C7-4KC are not covalently bond to the gold surface,
but they are only forming weak hydrogen bonds with the amino groups of the undesired PDL
on the surface. Unfortunately, the S 2p peak cannot be recorded due to the presence of the
shielding Si 2s peak, therefore the mixed SAM surface ratio cannot be calculated.
Glass substrates
The XPS analysis was performed on the glass substrates incubated in progesterone-C7-
4KC:EG6OH mixed SAMs, after the third step described before. The results of the analysis
revealed the presence of the elemental species C, N, O and Si. The C 1s spectrum (Figure 6.20
a) can be deconvoluted into three peaks, assignable to different binding environments334,335.
The first peak centred at 284.8 eV corresponds to C-C and C-Si bonds, the second peak centred
at 286.2 eV is ascribable to CONH groups, whereas the third peak at 288.0 eV is assigned to the
C 1s photoelectron of the carbonyl moiety, C=O. The N 1s spectrum (Figure 6.20 b) can be
deconvoluted into two peaks, the first one corresponding to amino (NH2) and amide groups
(CONH) at 399.5 eV and the second one ascribable to protonated amino groups (NH3+) at 400.4
eV383. The O 1s spectrum (Figure 6.20 c) is composed by two peaks, corresponding to three
CHAPTER 6 183
different binding environments334,335. The first peak at 531 eV corresponds to Si-OH moieties,
whereas the second peak centred at 532.5 eV can be assigned to C=O and Si-O-Si groups. The
Si 2p spectrum (Figure 6.20 d) can be deconvoluted into two peaks, the first one centred at
102.5 eV can be ascribed to Si-OH groups, whereas the second peak centred at 103.2 eV can
be assigned to SiOx present in the glass and Si-O-Si groups present in the silane
chains116,334,335,383–391. The silicon peaks recorded on glass were similar to those observed for
gold substrates after this last step, confirming that the formation of silane-PDL permanently
affects the surface, compromising the success of the orthogonal functionalisation strategy.
Figure 6.20 – XPS spectra of the a) C 1s, b) N 1s, c) O 1s and d) Si 2p regions after the
formation of Progesterone-C7-4KC:EG6OH mixed SAMs on glass substrates.
CHAPTER 6 184
However, in the region between 160.0 eV and 165.0 eV in the Si 2s spectrum, a small
peak is visible (Figure 6.21).
Figure 6.21 – XPS spectrum of Si 2s. A small peak is visible in the region of S 2p.
This peak could demonstrate the presence of the mixed SAMs on the glass substrates,
and we could hypothesise that it will compromise the sperm cell attachment on the glass
substrates. However, if we consider the background noise the peak could be questionable and
additional analysis needs to be done in the future. The progesterone present on glass
substrates could interact with sperm cells and trigger their hyperactivation236,240,381. This
phenomenon would make sperm cell to “swim away”, due to the hyper-motility acquired. To
verify this hypothesis, cell adhesion was tested on microscope glass slides after all four steps.
A deeper insight into the molecular coverage of the glass substrates can be obtained by
calculating the ratios between the elements analysed by XPS.
CHAPTER 6 185
The results evidence only a small increment of the nitrogen amount on the surface,
showing a variation in the N/C ratio from 0.132±0.001 to 0.19±0.01. This small change can be
due to the presence of some Progesterone-C7-4KC molecules on the surface. This, together
with the possible presence of a small S 2p in the silicon spectrum, does not exclude the
possibility of molecules of Progesterone-C7-4KC sitting on top of the silane-PDL layer,
interacting with poly-D-lysine side chains via hydrogen bonds between amino groups. The
interaction can be due to an insufficient percentage of triethylamine (TEA) in the mixed
monolayer, unable to prevent the formation of the undesired hydrogen bonds. The orthogonal
functionalisation investigated herein, as demonstrated by XPS, was not effective at promoting
the solely attachment of PDL on the glass surfaces and the Progesterone-C7-4KC:EG6OH mixed
SAMs on gold. Further evidence that further studies are needed in the future to optimise such
functionalization was acquired by conducting cell adhesion experiments.
6.3.4 Study of the effect of surface preparation steps on cell adhesion
After the completion of the fourth step of the surface preparation, the treated glass
slide was attached to the imaging chamber with vacuum grease and sperm cells were injected
into the chamber and left incubating following the procedure described in section 5.2.1.4. The
starting hypothesis is that the possible presence of Progesterone-C7-4KC on the glass surface
could restrain sperm cells from adhering. This adhesion hindrance could be due to the
interaction between the sperm cells and the progesterone. This interaction would trigger the
hyperactivation of the sperm cells that will therefore “swim-away” due to the hyper-motility
acquired and will be removed from the flow cell by vacuum suction. As expected from XPS
analysis results, the fluorescence microscopy analysis of the surface revealed no presence of
cells on the surface (Figure 6.22).
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Figure 6.22 – Fluorescence images of cell adhered on glass slides after the completion of the
Progesterone-C7-4KC:EG6OH mixed SAM deposition step.
The failure in sperm cells attachment can be related to the sulphur peak recorded on
the glass substrate, indicating the presence of progesterone-C7-4KC molecules, as seen in the
previous section.
6.4 Conclusions and Future Work
In conclusion, we have firstly studied sperm cells adhesion in different conditions. PDL
coating is the standard method used to promote cell adhesion312, but it resulted to be
incompatible with the conditions needed for the orthogonal functionalisation of the
micropattern that we aim to use in our future research work. We have successfully
circumvented this issue, by creating silane-PDL layers on glass. Amide bonds were formed,
between the carboxyl groups on silane molecules and amino groups on PDL, using different
concentrations of PDL (0.1 mg/ml and 0.5 mg/ml), via EDC/NHS coupling strategy. Labelled
sperm cells were then incubated on the different surfaces and the success of cell adhesion was
analysed by fluorescence microscopy. The silane-PDL layer formed using 0.5 mg/ml of PDL was
selected as the best one at promoting cell adhesion after overnight incubation in HPLC ethanol.
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An orthogonal self-assembly strategy was then designed, to create a device able to
promote sperm cells adhesion and control their hyperactivation in real-time. The detailed
analysis of the surfaces by XPS, after each functionalisation step, allowed us to investigate how
each step influences the performance of the next one. The information collected through the
XPS investigation was enormous and it will be useful to plan future work. The XPS analysis of
both clean glass and clean gold showed that working in a controlled environment (e.g. glove
box), is fundamental to avoid undesired contamination from organic compound present in the
laboratory environment. The functionalisation of glass surfaces with silane-PDL layers was
successful and it is not affected by the use of KOH aqueous solution for the removal of the
protective thiol. However, the silane and PDL molecules can contaminate the gold surfaces,
contributing to the failure of the removal of the 11-mercapto-1-undecanol monolayer. It will
also be important to investigate this step further, splitting it into two different analysis. Firstly,
the removal of 11-mercapto-1-undecanol monolayer should be investigated by XPS. Secondly,
the removal of the same monolayer should be attempted after the step involving the
deposition of silane-PDL molecules. The cyclic voltammogram should be then recorded to verify
that the curve corresponding to the thiol desorption is present. The investigation of the last
functionalisation step, involving the formation of progesterone-C7-4KC:EG6OH mixed SAM,
revealed the possible presence of progesterone-C7-4KC on the glass surface. As stated before,
this can lead to the impossibility of achieving a correct cell adhesion on glass surfaces. It will be
therefore worth testing different percentages of triethylamine (TEA) in the mixed SAM
solution, to find the optimum one that can prevent the formation of hydrogen bonds between
the amino groups of lysine groups on both PDL and progesterone-C7-4KC molecules. In
addition, each step can be tested again by XPS after sonicating the substrates, to investigate if
sonication could be beneficial to the functionalisation process. Finally, after the incubation of
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both glass and gold substrates in progesterone-C7-4KC:EG6OH solution, the XPS analysis
showed the incorrect formation of the mixed monolayer on gold substrates and the presence
of progesterone-C7-4KC molecules on top of silane-PDL layers on glass substrates. To gain a
better understanding of what is happening on the gold surfaces after the last step, SPR
experiments can be performed on these surfaces. The interaction between progesterone-Ab
and progesterone moieties can be tested in OC conditions to verify the presence of the desired
progesterone-C7-4KC:EG6OH mixed SAMs on the gold substrates. If the OC experiments are
successful, the feasibility of the switching can also be tested to model what we want to obtain
once the glass-gold micropattern has been correctly functionalised. Despite the difficulties
encountered in achieving the desired molecular system on the chosen substrates, orthogonal
functionalisation of surfaces remains an important tool in the development of miniaturised
devices possessing a wide range of applications. Such strategy allows the reduction in the use
of protective groups and number of reaction step, but also the use of mild conditions. This
study will certainly be useful in the development of new orthogonal chemistry strategies and
new tools applicable in both cell biology and the improvement of IVF techniques.
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Chapter 7 – Conclusions and Future Work
7.1 Conclusions
The research work described in this dissertation was aimed at the development of novel
biomolecular platforms able to control biointeractions upon the application of an electrical
potential.
Firstly, a switchable oligopeptide mixed SAM was investigated to analyse the possibility
of controlling the interaction between biotin end groups and neutravidin protein in solution.
The chosen mixed SAM was composed by biotin-4KC and triethylene glycol thiol molecules. The
4-Lys units gave the molecule a flexible and switchable backbone. In fact, lysine amino acids
present a positive charge a pH 7 that can be exploited to induce a molecular rearrangement on
the gold surface, upon the application of an electrical potential. The interaction between
neutravidin and biotin in different biotin-4KC:TEGT mixed SAM were monitored by
electrochemical SPR experiments to identify the mixed SAM ratio presenting the highest
switching ability.
The successful results obtained where then used to create a switchable platform to
control, for the first time, the interaction between an antigen on the surface and its antibody
in solution. Specifically, we studied the control of the interaction between progesterone and
anti-mouse progesterone antibodies. Different buffers were studied to identify the best
conditions for the switching.
Finally, preliminary studies were performed on glass and gold substrates to investigate
the feasibility of an orthogonal functionalisation of a micropatterned surface, with the aim of
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developing a novel smart molecular system able to expose and conceal progesterone moieties
on demand for the control of calcium signalling in sperm cells.
The attachment of sperm cells on different functionalised glass substrates were also
investigated.
Briefly:
Chapter 3 described the characterisation experiments and switching studies performed
on different biotin-4KC:TEGT ratios to identify the best surface organisation conditions needed
to have the best control over the interaction between biotin on the gold surface and
neutravidin in solution. Furthermore, the role of the oligopeptide chain was also investigated
to elucidate how the gap distance between the biotin end group and the ethylene glycol thiol
matrix can influence the dynamics of the switching process. The switching behaviour of two
different ratios of biotin-2KC:TEGT and biotin-6KC:TEGT mixed SAM were studied and compare
to the biotin-4KC:TEGT performance. An insight into the dynamics governing the molecular
rearrangement over the surface, when an electrical potential is applied, was obtained thanks
to molecular dynamics simulations performed by Nanjing University. The results showed that
the length of the oligopeptide chain has a fundamental role in the switching process and long
switchable chains can lead to intercrossing, reducing the switching ability of the mixed SAMs.
The matrix of ethylene glycol thiol has a fundamental role in preventing unspecific neutravidin
binding over the surface and in spacing out the oligopeptide chains to give them enough free
space to undergo the switching. In addition, it allows the embedding of the biotin active group
that becomes unavailable for binding to neutravidin. On the other hand, a well-packed ethylene
glycol thiol matrix can constrain the lysine chains from undergoing a molecular rearrangement
upon the application of an electrical potential.
CHAPTER 7 191
Chapter 4 illustrated the development of a switchable system for controlling antigen-
antibody interactions. Such a system was fabricated starting from the results obtained in
Chapter 3. Therefore, similar lysine oligopeptide chains were used to expose or conceal
molecules of progesterone on the gold surface, under potential control. After preliminary
studies in PBS, electrochemical SPR studies were performed on progesterone-C7-4KC:EG6OH
mixed SAMs, in different sEBSS+BSA buffer conditions, to investigate the effect of Bovine
Serum Albumin (BSA) on the mixed SAMs switching performance. The results showed that the
presence of a negatively charged protein such as BSA can hinder the correct switching of the
oligolysine chains. By removing BSA from the buffer, we were able to successfully exploit the
mixed monolayer to control, for the first time, the interaction between progesterone and anti-
mouse progesterone antibody.
Chapter 5 analysed, firstly, sperm cells adhesion in different conditions. A new adhesion
strategy, based on silane-PDL layers was investigated for the first time and compared to the
standard PDL coating method. The latter, resulted to be unsuitable for the orthogonal
functionalisation of glass-gold micropatterned surfaces that will be the one of the next steps of
this research work. An orthogonal surface functionalisation strategy was planned,
characterised and analysed, step by step, to find the possible inhibiting factors of the process.
The results showed that several factors need to be considered to achieve the desired and
correct molecular structure on both glass and gold surfaces. The number of possible and
necessary improvements of the strategy were described in detail in Chapter 5 conclusions
section. The most important factor to be taken into consideration is the possible cross
contamination of the two substrates, as a result of the functionalisation chemistry used.
CHAPTER 7 192
7.2 Future Work
The work performed in this thesis is the first effort in both controlling the interaction
between progesterone and the anti-mouse progesterone antibody and developing an
orthogonal functionalisation strategy to create a novel platform to control calcium signalling
activation in human sperm cells.
In this thesis, the factors that can influence the switching capability of different mixed
SAMs, such as SAM components surface ratio, switching units length and buffer conditions
were investigated. In addition, the analysis of the limitations affecting the feasibility of the
orthogonal functionalisation process, allowed the understanding of how the functionalisation
steps can influence each others and the data collected will be fundamental to plan out future
work.
Firstly, it will be important to study the switching ability of different progesterone-Cn-
4KC:EG6OH systems, to investigate the role of the alkyl spacer in the switching process. It would
also be interesting to investigate how the alkyl spacer length could influence the response of
sperm cells from different donors to the progesterone end group. In addition, different
antibody/antigen systems could be studied. For example, a different host could be used instead
of mouse (e.g. rabbit, goat) to produce monoclonal antibodies with different affinities to
progesterone. New SPR experiments in different electrical potential conditions can be
performed to compare the SPR response and the switching ability of the progesterone-
oligopeptide in the presence of different interacting partners in solution.
Moreover, the concept of using switchable peptide to conceal and expose larger
biomolecules on demand can be expanded, by investigating the application of nanobodies.398
Nanobodies are natural single-domain antibodies, which result to be particularly attractive as
CHAPTER 7 193
capturing molecules for use in biosensing. They recognise their antigens with high specificity
and affinities similar to standard antibodies (i.e. nano- to picomolar affinities), but thanks to
their small molecular size, they are able to recognise novel epitopes that regular-size antibodies
cannot.398,399 In fact, nanobodies exhibit a length down to 2-3 nm and molecular weight of
12-15 kDa, which are much smaller than those of antibodies (150-160 kDa).399
Additionally, different types of switching can be investigated. For example, future
research work can be aimed at the development of a double-armed molecule, exploiting
aspartic acid moieties as switching “arms”. Aspartic acid aminoacid present negatively-charged
side chains at pH 7. In this way, an OFF-ON switching can be studied, which will stop the need
to have an electrical potential to control the concealment of the bioactive molecule (Figure
7.1).
Figure 7.1 – Cartoon representation of double-armed switching molecule. The aspartic acid
oligopeptide arms (green) are connected to the alkyl chain (black) carrying the progesterone
moiety (red) through a core central molecule (blue) in a dendron-like structure.
CHAPTER 7 194
Additional research interest can also be directed to the study of more rigid charged
backbones, such as phosphate backbones, instead of lysine-oligopeptides ones.
Furthermore, it will be fundamental to analyse some of the orthogonal functionalisation
steps in more detail, such as studying if sonicating the substrates could have any effect and if
changing the percentage of triethylamine (TEA) in the last step, could reduce the formation of
hydrogen bonds between lysine side chains.
The switchable mixed SAMs can also be modified and functionalised with different
antigens and find an important application in biomedicine, in the study of pathologies involving
antibodies.
Reversibility is still an issue and future research focus can be directed to the design of
new molecular structures that will allow the formation of a fully reversible system. Also, the
long-term stability of switching oligopeptides in complex biological conditions still need to be
investigated.
Once all the issues encountered in this research work will have been addressed and
solved, it will be possible to develop and analyse an innovative platform, able to control the
hyperactivation of human sperm cells, upon the application of an electrical potential. The
results of these future studies will be then applied to the selection of “healthy” sperm cells for
in-vitro fertilisation (IVF) techniques, increasing their success rates and reducing their costs.
REFERENCES 195
References
(1) Mendes, P. M. Chem. Soc. Rev. 2013, 42 (24), 9207–9218.
(2) Kasemo, B. Surf. Sci. 2002, 500 (1–3), 656–677.
(3) Chen, A.; Chatterjee, S. Chem. Soc. Rev. 2013, 42 (12), 5425.
(4) Mendes, P. M.; Yeung, C. L.; Preece, J. A. Nanoscale Res. Lett. 2007, 2 (8), 373–384.
(5) Wang, X.; Liu, L.-H.; Ramstrom, O.; Yan, M. Exp. Biol. Med. 2009, 234 (10), 1128–1139.
(6) Brolo, A. Nat. Photonics 2012, 6 (November), 709–713.
(7) Duke, C. B.; Plummer, E. W. 2002, i, 0–1.
(8) Wang, X.; Hu, Y.; Wei, H. Inorg. Chem. Front. 2016, 3 (1), 41–60.
(9) Nandivada, H.; Ross, A. M.; Lahann, J. Prog. Polym. Sci. 2010, 35 (1–2), 141–154.
(10) Pranzetti, A.; Preece, J. A.; Mendes, P. M. In Intelligent Stimuli-Responsive Materials;
John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2013; pp 377–422.
(11) Sin, M. L.; Mach, K. E.; Wong, P. K.; Liao, J. C. Expert Rev. Mol. Diagn. 2014, 14 (2), 225–
244.
(12) Tothill, I. E. Semin. Cell Dev. Biol. 2009, 20 (1), 55–62.
(13) Soper, S. A.; Brown, K.; Ellington, A.; Frazier, B.; Garcia-Manero, G.; Gau, V.; Gutman, S.
I.; Hayes, D. F.; Korte, B.; Landers, J. L.; Larson, D.; Ligler, F.; Majumdar, A.; Mascini, M.;
Nolte, D.; Rosenzweig, Z.; Wang, J.; Wilson, D. Biosens. Bioelectron. 2006, 21 (10),
1932–1942.
(14) Rasooly, A.; Jacobson, J. Biosens. Bioelectron. 2006, 21 (10), 1851–1858.
(15) Søndergaard, R. V.; Christensen, N. M.; Henriksen, J. R.; Kumar, E. K. P.; Almdal, K.;
Andresen, T. L. Chem. Rev. 2015, 115 (16), 8344–8378.
(16) Holzinger, M.; Le Goff, A.; Cosnier, S. Front. Chem. 2014, 2.
REFERENCES 196
(17) Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. Chem. Rev. 2014, 114 (15), 7421–7441.
(18) Razavi, H.; Janfaza, S. Nanomed J 2015, 2 (2), 74–87.
(19) Taleat, Z.; Khoshroo, A.; Mazloum-Ardakani, M. Microchim. Acta 2014, 181 (9–10),
865–891.
(20) Bauch, M.; Toma, K.; Toma, M.; Zhang, Q.; Dostalek, J. Plasmonics 2014, 9 (4), 781–799.
(21) Liu, X.; Li, H.; Jin, Q.; Ji, J. Small 2014, n/a-n/a.
(22) Ravindran, A.; Chandran, P.; Khan, S. S. Colloids Surfaces B Biointerfaces 2013, 105,
342–352.
(23) Omidfar, K.; Khorsand, F.; Darziani Azizi, M. Biosens. Bioelectron. 2013, 43, 336–347.
(24) Zhang, X. Cell Biochem. Biophys. 2015, 72 (3), 771–775.
(25) Yang, N.; Chen, X.; Ren, T.; Zhang, P.; Yang, D. Sensors Actuators B Chem. 2015, 207,
690–715.
(26) Kumar, S.; Ahlawat, W.; Kumar, R.; Dilbaghi, N. Biosens. Bioelectron. 2015, 70, 498–
503.
(27) Gaharwar, A. K.; Peppas, N. A.; Khademhosseini, A. Biotechnol. Bioeng. 2014, 111 (3),
441–453.
(28) Cirillo, G.; Hampel, S.; Spizzirri, U. G.; Parisi, O. I.; Picci, N.; Iemma, F. Biomed Res. Int.
2014, 2014, 1–17.
(29) Wong, B. S.; Yoong, S. L.; Jagusiak, A.; Panczyk, T.; Ho, H. K.; Ang, W. H.; Pastorin, G.
Adv. Drug Deliv. Rev. 2013, 65 (15), 1964–2015.
(30) Mehra, N. K.; Jain, K.; Jain, N. K. Drug Discov. Today 2015, 20 (6), 750–759.
(31) Sajid, M. I.; Jamshaid, U.; Jamshaid, T.; Zafar, N.; Fessi, H.; Elaissari, A. Int. J. Pharm.
2016, 501 (1–2), 278–299.
(32) Ferreira, P.; Alves, P.; Coimbra, P.; Gil, M. H. J. Coatings Technol. Res. 2015, 12 (3), 463–
REFERENCES 197
475.
(33) Crespilho, F. N.; Zucolotto, V.; Oliveira Jr., O. N.; Nart, F. C. Int. J. Electrochem. Sci. 2006,
1 (September), 194–214.
(34) Yost, A. L.; Shahsavari, S.; Bradwell, G. M.; Polak, R.; Fachin, F.; Cohen, R. E.; McKinley,
G. H.; Toner, M.; Rubner, M. F.; Wardle, B. L. Microsystems Nanoeng. 2015, 1 (0),
15037.
(35) Schreiber, F. Prog. Surf. Sci. 2000, 65 (5–8), 151–256.
(36) Ferretti, S.; Paynter, S.; Russell, D. A.; Sapsford, K. E.; Richardson, D. J. TrAC Trends
Anal. Chem. 2000, 19 (9), 530–540.
(37) Senaratne, W.; Andruzzi, L.; Ober, C. K.; Monolayers, S.-A.; Perspectives, F.
Biomacromolecules 2005, 6 (5), 2427–2448.
(38) Schreiber, F. J. Phys. Condens. Matter 2004, 16 (4), 881–900.
(39) Sung, W. C.; Chang, C. C.; Makamba, H.; Chen, S. H. Anal. Chem. 2008, 80 (5), 1529–
1535.
(40) Yang, M.; Choi, D.; Choi, M.; Hong, J. J. Ind. Eng. Chem. 2016, 33, 221–225.
(41) Dai, Z.; Ju, H. TrAC - Trends Anal. Chem. 2012, 39, 149–162.
(42) Santos, A.; Kumeria, T.; Losic, D. TrAC - Trends Anal. Chem. 2013, 44, 25–38.
(43) Wei, H.; Wang, E. Chem. Soc. Rev. 2013, 42 (14), 6060–6093.
(44) Wang, X.; Hu, Y.; Wei, H. Inorg. Chem. Front. 2016, 3 (1), 41–60.
(45) Peppas, N. A.; Van Blarcom, D. S. J. Control. Release 2015.
(46) Chik, H.; Xu, J. M. Mater. Sci. Eng. R Reports 2004, 43 (4), 103–138.
(47) Ghicov, A.; Schmuki, P. Chem. Commun. (Camb). 2009, No. 20, 2791–2808.
(48) Wang, Z.; Liu, H.; Yang, S. H.; Wang, T.; Liu, C.; Cao, Y. C. Proc. Natl. Acad. Sci. 2012, 109
(31), 12387–12392.
REFERENCES 198
(49) Dugan, L. L.; Lovett, E. G.; Quick, K. L.; Lotharius, J.; Lin, T. T.; O’Malley, K. L.
Parkinsonism Relat. Disord. 2001, 7 (3), 243–246.
(50) Yao, W.-T.; Zhu, H.-Z.; Li, W.-G.; Yao, H.-B.; Wu, Y.-C.; Yu, S.-H. Chempluschem 2013, 78
(7), 723–727.
(51) Qu, K.; Shi, P.; Ren, J.; Qu, X. Chem. - A Eur. J. 2014, 20 (24), 7501–7506.
(52) Li, N.; Yan, Y.; Xia, B.-Y.; Wang, J.-Y.; Wang, X. Biosens. Bioelectron. 2014, 54, 521–527.
(53) Asati, A.; Santra, S.; Kaittanis, C.; Nath, S.; Perez, J. M. Angew. Chemie Int. Ed. 2009, 48
(13), 2308–2312.
(54) Pirmohamed, T.; Dowding, J. M.; Singh, S.; Wasserman, B.; Heckert, E.; Karakoti, A. S.;
King, J. E. S.; Seal, S.; Self, W. T. Chem. Commun. 2010, 46 (16), 2736.
(55) He, W.; Liu, Y.; Yuan, J.; Yin, J. J.; Wu, X.; Hu, X.; Zhang, K.; Liu, J.; Chen, C.; Ji, Y.; Guo, Y.
Biomaterials 2011, 32 (4), 1139–1147.
(56) Wei, H.; Wang, E. Anal. Chem. 2008, 80 (6), 2250–2254.
(57) Hoffman, A. S. Adv. Drug Deliv. Rev. 2012, 64, 18–23.
(58) Kuwana, E.; Liang, F.; Sevick-Muraca, E. M. Biotechnol. Prog. 2004, 20 (5), 1561–1566.
(59) Mader, H. S.; Wolfbeis, O. S. Microchim. Acta 2008, 162 (1–2), 1–34.
(60) Biswas, A.; Bayer, I. S.; Biris, A. S.; Wang, T.; Dervishi, E.; Faupel, F. Adv. Colloid
Interface Sci. 2012, 170 (1–2), 2–27.
(61) Pen, P. E. N. .
(62) Wu, C. C.; Reinhoudt, D. N.; Otto, C.; Subramaniam, V.; Velders, A. H. Small 2011, 7 (8),
989–1002.
(63) Koegler, P.; Clayton, A.; Thissen, H.; Santos, G. N. C.; Kingshott, P. Adv. Drug Deliv. Rev.
2012, 64 (15), 1820–1839.
(64) Jung, G. Y.; Li, Z.; Wu, W.; Chen, Y.; Olynick, D. L.; Wang, S. Y.; Tong, W. M.; Williams, R.
REFERENCES 199
S. Langmuir 2005, 21 (4), 1158–1161.
(65) Yu, Y.; Zhang, G. Updat. Adv. Lithogr. 2013, 3–34.
(66) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D. E. Annu. Rev. Biomed.
Eng. 2001, 3 (1), 335–373.
(67) Zhang, J.; Yang, B. Adv. Funct. Mater. 2010, 20 (20), 3411–3424.
(68) Vieu, C.; Carcenac, F.; Pepin, A.; Chen, Y.; Mejias, M.; Lebib, A.; Manin-Ferlazzo, L.;
Couraud, L.; Launois, H. Appl. Surf. Sci. 2000, 164, 111–117.
(69) Manfrinato, V. R.; Zhang, L.; Su, D.; Duan, H.; Hobbs, R. G.; Stach, E. A.; Berggren, K. K.
Nano Lett. 2013, 13 (4), 1555–1558.
(70) Xia, Y.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153–184.
(71) Xia, Y.; Rogers, J. A.; Paul, K. E.; Whitesides, G. M. Chem. Rev. 1999, 99 (7), 1823–1848.
(72) Yang, S. M.; Jang, S. G.; Choi, D. G.; Kim, S.; Yu, H. K. Small 2006, 2 (4), 458–475.
(73) Wood, M. a. J. R. Soc. Interface 2007, 4 (12), 1–17.
(74) Shimomura, M.; Sawadaishi, T. Curr. Opin. Colloid Interface Sci. 2001, 6 (1), 11–16.
(75) Borges, J.; Mano, J. F. Chem. Rev. 2014, 114 (18), 8883–8942.
(76) Langmuir, I.; Schaefer, V. J. J. Am. Chem. Soc. 1937, 59 (10), 2075–2076.
(77) Blodgett, K. B. J. Am. Chem. Soc. 1935, 57 (6), 1007–1022.
(78) Ulman, a. Chem. Rev. 1996, 96 (4), 1533–1554.
(79) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111 (18), 7155–7164.
(80) Laibinis, P. E.; Nuzzo, R. G.; Whitesides, G. M. J. Phys. Chem. 1992, 96 (12), 5097–5105.
(81) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111 (18), 7155–7164.
(82) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-
Assembly; Academic Press, 2013.
(83) Bishop, A. R.; Nuzzo, R. G. Curr. Opin. Colloid Interface Sci. 1996, 1 (1), 127.
REFERENCES 200
(84) Bigelow, W. C.; Pickett, D. L.; Zisman, W. A. J. Colloid Sci. 1946, 1 (6), 513–538.
(85) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105 (13), 4481–4483.
(86) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Self-assembled
monolayers of thiolates on metals as a form of nanotechnology; 2005; Vol. 105.
(87) Vericat, C.; Vela, M. E.; Benitez, G.; Carro, P.; Salvarezza, R. C. Chem. Soc. Rev. 2010, 39
(5), 1805.
(88) Lee, S.; Puck, A.; Graupe, M.; Colorado, R.; Shon, Y. S.; Lee, T. R.; Perry, S. S. Langmuir
2001, 17 (23), 7364–7370.
(89) Yan, C.; Zharnikov, M.; Gölzhäuser, A.; Grunze, M. Langmuir 2000, 16 (15), 6208–6215.
(90) Crego-Calama, M.; Reinhoudt, D. N. Adv. Mater. 2001, 13 (15), 1171–1174.
(91) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Phys. Org. Chem. 2001, 14 (7), 407–
415.
(92) Herzer, N.; Hoeppener, S.; Schubert, U. S. Chem. Commun. (Camb). 2010, 46 (31),
5634–5652.
(93) Onclin, S.; Ravoo, B. J.; Reinhoudt, D. N. Angew. Chemie Int. Ed. 2005, 44 (39), 6282–
6304.
(94) Badia, A.; Singh, S.; Demers, L.; Cuccia, L.; Brown, G. R.; Lennox, R. B. Chem. - A Eur. J.
1996, 2 (3), 359–363.
(95) Lenahan, K. M.; Wang, Y.-X.; Liu, Y.; Claus, R. O.; Heflin, J. R.; Marciu, D.; Figura, C. Adv.
Mater. 1998, 10 (11), 853–855.
(96) Chandross, M.; Lorenz, C. D.; Grest, G. S.; Stevens, M. J.; Webb, E. B. JOM 2005, 57 (9),
55–61.
(97) Petrenko, V. F.; Peng, S. Can. J. Phys. 2003, 81 (1–2), 387–393.
(98) Chaki, N. K.; Vijayamohanan, K. Biosens. Bioelectron. 2002, 17 (1–2), 1–12.
REFERENCES 201
(99) Guo, Y.; Li, M.; Mylonakis, A.; Han, J.; MacDiarmid, A. G.; Chen, X.; Lelkes, P. I.; Wei, Y.
Biomacromolecules 2007, 8 (10), 3025–3034.
(100) Inaba, R.; Khademhosseini, A.; Suzuki, H.; Fukuda, J. Biomaterials 2009, 30 (21), 3573–
3579.
(101) Sharma, H.; Nguyen, D.; Chen, A.; Lew, V.; Khine, M. Ann. Biomed. Eng. 2011, 39 (4),
1313–1327.
(102) Kumar, S.; Kumar, S.; Ali, M. A.; Anand, P.; Agrawal, V. V.; John, R.; Maji, S.; Malhotra,
B. D. Biotechnol. J. 2013, 8 (11), 1267–1279.
(103) Laibinis, P. E.; Fox, M. A.; Folkers, J. P.; Whitesides, G. M. Langmuir 1991, 7 (12), 3167.
(104) Lessel, M.; Bäumchen, O.; Klos, M.; Hähl, H.; Fetzer, R.; Paulus, M.; Seemann, R.;
Jacobs, K. Surf. Interface Anal. 2015, 47 (5), 557–564.
(105) MCGOVERN, M. E.; THOMPSON, M. Anal. Commun. 1998, 35 (12), 391–393.
(106) Smith, R. K.; Lewis, P. A.; Weiss, P. S. Prog. Surf. Sci. 2004, 75 (1–2), 1–68.
(107) Garg, N.; Carrasquillo-Molina, E.; Lee, T. R. Langmuir 2002, 18 (7), 2717–2726.
(108) Guo, L.-Y.; Zhao, Y.-P. J. Adhes. Sci. Technol. 2006, 20 (12), 1281–1293.
(109) Sagiv, J. J. Am. Chem. Soc. 1980, 399 (1976), 92–98.
(110) Parikh, A. N.; Allara, D. L.; Azouz, I. Ben; Rondelez, F. J. Phys. Chem. 1994, 98 (31),
7577–7590.
(111) Haensch, C.; Hoeppener, S.; Schubert, U. S. Chem. Soc. Rev. 2010, 39 (6), 2323.
(112) Schwartz, D. K. Annu. Rev. Phys. Chem. 2001, 52 (1), 107–137.
(113) Wouters, D.; Hoeppener, S.; Sturms, J. P. E.; Schubert, U. S. J. Scanning Probe Microsc.
2006, 1 (1), 45–50.
(114) Sun, L. J. Electrochem. Soc. 1991, 138 (8), L23.
(115) Menzel, H.; Stamm, M.; Heise, A.; Duschner, H.; Rauscher, M. Thin Solid Films 1998,
REFERENCES 202
327–329, 199–203.
(116) Chandekar, A.; Sengupta, S. K.; Whitten, J. E. Appl. Surf. Sci. 2010, 256 (9), 2742–2749.
(117) Wang, M.; Liechti, K. M.; Wang, Q.; White, J. M. Langmuir 2005, 21 (5), 1848–1857.
(118) Henderson, A. P.; Seetohul, L. N.; Dean, A. K.; Russell, P.; Pruneanu, S.; Ali, Z. Langmuir
2009, 25 (2), 931–938.
(119) Bürgi, T. Nanoscale 2015, 7 (38), 15553–15567.
(120) Alves, C. A.; Smith, E. L.; Porter, M. D. J. Am. Chem. Soc. 1992, 114 (4), 1222–1227.
(121) Tolstyka, Z. P.; Richardson, W.; Bat, E.; Stevens, C. J.; Parra, D. P.; Dozier, J. K.;
Distefano, M. D.; Dunn, B.; Maynard, H. D. ChemBioChem 2013, 14 (18), 2464–2471.
(122) Wilbur James, L.; Kumar, A.; Biebuyck, H. A. 1996, 452.
(123) Kang, J. F.; Liao, S.; Jordan, R.; Ulman, A. J. Am. Chem. Soc. 1998, 120 (37), 9662–9667.
(124) Ballav, N.; Terfort, A.; Zharnikov, M. Langmuir 2009, 25 (16), 9189–9196.
(125) Jeuken, L. J. C.; Daskalakis, N. N.; Han, X.; Sheikh, K.; Erbe, A.; Bushby, R. J.; Evans, S. D.
Sensors Actuators, B Chem. 2007, 124 (2), 501–509.
(126) Stranick, S. J.; Parikh, A. N.; Tao, Y.-T.; Allara, D. L.; Weiss, P. S. J. Phys. Chem. 1994, 98
(31), 7636–7646.
(127) Choi, I.; Kim, Y.; Kang, S. K.; Lee, J.; Yi, J. Langmuir 2006, 22 (11), 4885–4889.
(128) Jeuken, L. J. C.; Daskalakis, N. N.; Han, X.; Sheikh, K.; Erbe, A.; Bushby, R. J.; Evans, S. D.
Sensors Actuators B Chem. 2007, 124 (2), 501–509.
(129) Yaliraki, S. N.; Longo, G.; Gale, E.; Szleifer, I.; Ratner, M. A. J. Chem. Phys. 2006, 125 (7),
74708.
(130) Cossaro, A.; Mazzarello, R.; Rousseau, R.; Casalis, L.; Verdini, A.; Kohlmeyer, A.;
Floreano, L.; Scandolo, S.; Morgante, A.; Klein, M. L.; Scoles, G. Science (80-. ). 2008,
321 (5891), 943–946.
REFERENCES 203
(131) Smith, R. K.; Reed, S. M.; Lewis, P. A.; Monnell, J. D.; Clegg, R. S.; Kelly, K. F.; Bumm, L.
A.; Hutchison, J. E.; Weiss, P. S. J. Phys. Chem. B 2001, 105 (6), 1119–1122.
(132) McCarley, R. L.; Dunaway, D. J.; Willicut, R. J. Langmuir 1993, 9 (11), 2775–2777.
(133) Wirth, M. J. Science (80-. ). 1997, 275 (5296), 44–47.
(134) Senaratne, W.; Andruzzi, L.; Ober, C. K. Biomacromolecules 2005, 6 (5), 2427–2448.
(135) Hays, H. C. W.; Millner, P. A.; Prodromidis, M. I. Sensors Actuators B Chem. 2006, 114
(2), 1064–1070.
(136) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. J.
Am. Chem. Soc. 2000, 122 (34), 8303–8304.
(137) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115 (23), 10714–10721.
(138) Arima, Y.; Iwata, H. Biomaterials 2007, 28 (20), 3074–3082.
(139) Heuberger, R.; Sukhorukov, G.; Vörös, J.; Textor, M.; Möhwald, H. Adv. Funct. Mater.
2005, 15 (3), 357–366.
(140) Zhu, B.; Eurell, T.; Gunawan, R.; Leckband, D. J. Biomed. Mater. Res. 2001, 56 (3), 406–
416.
(141) Zheng, J.; Li, L.; Tsao, H.-K.; Sheng, Y.-J.; Chen, S.; Jiang, S. Biophys. J. 2005, 89 (1), 158–
166.
(142) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B
1998, 102 (2), 426–436.
(143) Diamandis, E. P.; Christopoulos, T. K. Clin. Chem. 1991, 37 (5), 625–636.
(144) Bu, D.; Zhuang, H.; Zhou, X.; Yang, G. Talanta 2014, 120, 40–46.
(145) Lei, K. F.; Yang, S.-I.; Tsai, S.-W.; Hsu, H.-T. Talanta 2015, 134, 264–270.
(146) Kendall, C.; Ionescu-Matiu, I.; Dreesman, G. R. J. Immunol. Methods 1983, 56 (3), 329–
339.
REFERENCES 204
(147) Chen, Y. P.; Zou, M. qiang; Qi, C.; Xie, M.-X.; Wang, D.-N.; Wang, Y.-F.; Xue, Q.; Li, J.-F.;
Chen, Y. Biosens. Bioelectron. 2013, 39 (1), 112–117.
(148) Focsan, M.; Campu, A.; Craciun, A.-M.; Potara, M.; Leordean, C.; Maniu, D.; Astilean, S.
Biosens. Bioelectron. 2016, 86, 728–735.
(149) Kim, M.-C.; Hong, M.-H.; Lee, B.-H.; Choi, H.-J.; Ko, Y.-M.; Lee, Y.-K. Ann. Biomed. Eng.
2015, 43 (12), 3004–3014.
(150) Tallawi, M.; Rosellini, E.; Barbani, N.; Cascone, M. G.; Rai, R.; Saint-Pierre, G.;
Boccaccini, A. R. J. R. Soc. Interface 2015, 12 (108), 20150254.
(151) Biju, V. Chem. Soc. Rev. 2014, 43 (3), 744–764.
(152) Patra, D.; Sengupta, S.; Duan, W.; Zhang, H.; Pavlick, R.; Sen, A. Nanoscale 2013, 5 (4),
1273–1283.
(153) Green, N. M. In Advances in Protein Chemistry; Elsevier Inc., 1975; pp 85–133.
(154) Piran, U.; Riordan, W. J. J. Immunol. Methods 1990, 133 (1), 141–143.
(155) Morgan, H.; Taylor, D. M. Biosens. Bioelectron. 1992, 7 (6), 405–410.
(156) Zimbron, J. M.; Heinisch, T.; Schmid, M.; Hamels, D.; Nogueira, E. S.; Schirmer, T.;
Ward, T. R. J. Am. Chem. Soc. 2013, 135 (14), 5384–5388.
(157) Yue, M.; Stachowiak, J. C.; Lin, H.; Datar, R.; Cote, R.; Majumdar, A. Nano Lett. 2008, 8
(2), 520–524.
(158) Holford, T. R. J.; Davis, F.; Higson, S. P. J. Biosens. Bioelectron. 2012, 34 (1), 12–24.
(159) Wolny, P. M.; Spatz, J. P.; Richter, R. P. Langmuir 2010, 26 (2), 1029–1034.
(160) DeLange, R. J.; Huang, T.-S. J. Biol. Chem. 1971, 246 (3), 698–709.
(161) Holford, T. R. J.; Davis, F.; Higson, S. P. J. Biosens. Bioelectron. 2012, 34 (1), 12–24.
(162) Malmqvist, M. Curr. Opin. Immunol. 1993, 5 (2), 282–286.
(163) Byrne, B.; Stack, E.; Gilmartin, N.; O’Kennedy, R. Sensors 2009, 9 (6), 4407–4445.
REFERENCES 205
(164) Blake, D. A.; Jones, R. M.; Blake, R. C.; Pavlov, A. R.; Darwish, I. A.; Yu, H. Biosens.
Bioelectron. 2001, 16 (9–12), 799–809.
(165) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chemie 2005, 117 (34), 5592–
5595.
(166) Kirby, R.; Cho, E. J.; Gehrke, B.; Bayer, T.; Park, Y. S.; Neikirk, D. P.; McDevitt, J. T.;
Ellington, A. D. Anal. Chem. 2004, 76 (14), 4066–4075.
(167) Karlsson, R.; Michaelsson, A.; Mattsson, L. J. Immunol. Methods 1991, 145 (1–2), 229–
240.
(168) Ashley, J.; Piekarska, M.; Segers, C.; Trinh, L.; Rodgers, T.; Willey, R.; Tothill, I. E.
Biosens. Bioelectron. 2017, 88, 109–113.
(169) Buchatip, S.; Ananthanawat, C.; Sithigorngul, P.; Sangvanich, P.; Rengpipat, S.; Hoven,
V. P. Sensors Actuators B Chem. 2010, 145 (1), 259–264.
(170) Wang, W.; Singh, S.; Zeng, D. L.; King, K.; Nema, S. J. Pharm. Sci. 2007, 96 (1), 1–26.
(171) Leenaars, M.; Hendriksen, C. F. M. ILAR J. 2005, 46 (3), 269–279.
(172) Li, J.; Sun, Q.; Han, S.; Wang, J.; Wang, Z.; Jin, C. Prog. Org. Coatings 2015, 87, 155–160.
(173) Mendes, P. M. Chem. Soc. Rev. 2008, 37 (11), 2512.
(174) Cantini, E.; Wang, X.; Koelsch, P.; Preece, J. A.; Ma, J.; Mendes, P. M. Acc. Chem. Res.
2016, 49 (6), 1223–1231.
(175) Ebara, M. .; Kotsuchibashi, Y. .; Narain, R. .; Idota, N. .; Kim, Y. .; Hoffman, J. M. .; Uto, K.
Smart Biomaterials; Springer, 2014.
(176) Yamato, M.; Utsumi, M.; Kushida, A.; Konno, C.; Kikuchi, A.; Okano, T. Tissue Eng. 2001,
7 (4), 473–480.
(177) Cammas, S.; Suzuki, K.; Sone, C.; Sakurai, Y.; Kataoka, K.; Okano, T. J. Control. Release
1997, 48 (2–3), 157–164.
REFERENCES 206
(178) Takezawa, T.; Mori, Y.; Yoshizato, K. Bio/Technology 1990, 8 (9), 854–856.
(179) Schmaljohann, D. Adv. Drug Deliv. Rev. 2006, 58 (15), 1655–1670.
(180) Jonas, A. M.; Glinel, K.; Oren, R.; Nysten, B.; Huck, W. T. S. Macromolecules 2007, 40
(13), 4403–4405.
(181) Zhang, X.-Z.; Zhuo, R.-X.; Cui, J.-Z.; Zhang, J.-T. Int. J. Pharm. 2002, 235 (1–2), 43–50.
(182) Schmaljohann, D.; Oswald, J.; Jørgensen, B.; Nitschke, M.; Beyerlein, D.; Werner, C.
Biomacromolecules 2003, 4 (6), 1733–1739.
(183) Liu, F.; Urban, M. W. Prog. Polym. Sci. 2010, 35 (1–2), 3–23.
(184) Xia, F.; Zhu, Y.; Feng, L.; Jiang, L. Soft Matter 2009, 5 (2), 275–281.
(185) Jiang, W.; Wang, G.; He, Y.; Wang, X.; An, Y.; Song, Y.; Jiang, L. Chem. Commun. 2005,
No. 28, 3550.
(186) Yu, X.; Wang, Z.; Jiang, Y.; Shi, F.; Zhang, X. Adv. Mater. 2005, 17 (10), 1289–1293.
(187) Xia, F.; Feng, L.; Wang, S.; Sun, T.; Song, W.; Jiang, W.; Jiang, L. Adv. Mater. 2006, 18 (4),
432–436.
(188) Park, C.; Oh, K.; Lee, S. C.; Kim, C. Angew. Chemie Int. Ed. 2007, 46 (9), 1455–1457.
(189) Chen, Y.-C.; Xie, R.; Chu, L.-Y. J. Memb. Sci. 2013, 442, 206–215.
(190) Gawel, K.; Barriet, D.; Sletmoen, M.; Stokke, B. T. Sensors 2010, 10 (5), 4381–4409.
(191) Li, P.-F.; Xie, R.; Fan, H.; Ju, X.-J.; Chen, Y.-C.; Meng, T.; Chu, L.-Y. Ind. Eng. Chem. Res.
2012, 51 (28), 9554–9563.
(192) Czaun, M.; Hevesi, L.; Takafuji, M.; Ihara, H. Chem. Commun. 2008, No. 18, 2124.
(193) Woodward, R. T.; Olariu, C. I.; Hasan, E. A.; Yiu, H. H. P.; Rosseinsky, M. J.; Weaver, J. V.
M. Soft Matter 2011, 7 (9), 4335.
(194) Pranzetti, A.; Davis, M.; Yeung, C. L.; Preece, J. A.; Koelsch, P.; Mendes, P. M. Adv.
Mater. Interfaces 2014, 1 (5), 1400026.
REFERENCES 207
(195) Pranzetti, A.; Mieszkin, S.; Iqbal, P.; Rawson, F. J.; Callow, M. E.; Callow, J. A.; Koelsch,
P.; Preece, J. A.; Mendes, P. M. Adv. Mater. 2013, 25 (15), 2181–2185.
(196) Yeung, C. L.; Iqbal, P.; Allan, M.; Lashkor, M.; Preece, J. A.; Mendes, P. M. Adv. Funct.
Mater. 2010, 20 (16), 2657–2663.
(197) Yeung, C. L.; Wang, X.; Lashkor, M.; Cantini, E.; Rawson, F. J.; Iqbal, P.; Preece, J. A.; Ma,
J.; Mendes, P. M. Adv. Mater. Interfaces 2014, 1 (2).
(198) Lashkor, M.; Rawson, F. J.; Preece, J. A.; Mendes, P. M. Analyst 2014, 139 (21), 5400–
5408.
(199) Lashkor, M.; Rawson, F. J.; Stephenson-Brown, A.; Preece, J. A.; Mendes, P. M. Chem.
Commun. 2014, 50 (98), 15589–15592.
(200) Krishnamoorthy, M.; Hakobyan, S.; Ramstedt, M.; Gautrot, J. E. Chem. Rev. 2014, 114
(21), 10976–11026.
(201) Schmidt, S.; Zeiser, M.; Hellweg, T.; Duschl, C.; Fery, A.; Möhwald, H. Adv. Funct. Mater.
2010, 20 (19), 3235–3243.
(202) Wischerhoff, E.; Uhlig, K.; Lankenau, A.; Börner, H. G.; Laschewsky, A.; Duschl, C.; Lutz,
J. Angew. Chemie Int. Ed. 2008, 47 (30), 5666–5668.
(203) Synytska, A.; Svetushkina, E.; Puretskiy, N.; Stoychev, G.; Berger, S.; Ionov, L.; Bellmann,
C.; Eichhorn, K.-J.; Stamm, M. Soft Matter 2010, 6 (23), 5907.
(204) Flink, S.; Veggel, F. C. J. M. van; Reinhoudt, D. N. Adv. Mater. 2000, 12 (18), 1315–1328.
(205) Lahann, J. Science (80-. ). 2003, 299 (5605), 371–374.
(206) Liu, Y.; Mu, L.; Liu, B.; Zhang, S.; Yang, P.; Kong, J. Chem. Commun. 2004, No. 10, 1194.
(207) Liu, F.; Urban, M. W. Prog. Polym. Sci. 2010, 35 (1–2), 3–23.
(208) Mendes, P. M.; Christman, K. L.; Parthasarathy, P.; Schopf, E.; Ouyang, J.; Yang, Y.;
Preece, J. A.; Maynard, H. D.; Chen, Y.; Stoddart, J. F. Bioconjug. Chem. 2007, 18 (6),
REFERENCES 208
1919–1923.
(209) Liu, Y.; Mu, L.; Liu, B.; Kong, J. Chem. - A Eur. J. 2005, 11 (9), 2622–2631.
(210) Rant, U.; Arinaga, K.; Scherer, S.; Pringsheim, E.; Fujita, S.; Yokoyama, N.; Tornow, M.;
Abstreiter, G. Proc. Natl. Acad. Sci. 2007, 104 (44), 17364–17369.
(211) Doyle, D. A. Science (80-. ). 1998, 280 (5360), 69–77.
(212) Catterall, W. A. Exp. Physiol. 2014, 99 (1), 35–51.
(213) Knezevic, J.; Langer, A.; Hampel, P. A.; Kaiser, W.; Strasser, R.; Rant, U. J. Am. Chem.
Soc. 2012, 134 (37), 15225–15228.
(214) de Kretser DM1. Lancet 1997, 349 ((9054)), 787–790.
(215) Brugh, V. M.; Lipshultz, L. I. Med. Clin. North Am. 2004, 88 (2), 367–385.
(216) Cooper, T. G.; Noonan, E.; von Eckardstein, S.; Auger, J.; Baker, H. W. G.; Behre, H. M.;
Haugen, T. B.; Kruger, T.; Wang, C.; Mbizvo, M. T.; Vogelsong, K. M. Hum. Reprod.
Update 2010, 16 (3), 231–245.
(217) Moskovtsev, S. I.; Willis, J.; Mullen, J. B. M. Fertil. Steril. 2006, 85 (2), 496–499.
(218) Sharpe, R. M. Best Pract. Res. Clin. Endocrinol. Metab. 2000, 14 (3), 489–503.
(219) Oliva, A. Hum. Reprod. 2001, 16 (8), 1768–1776.
(220) Tremellen, K. Hum. Reprod. Update 2008, 14 (3), 243–258.
(221) Bozhedomov, V. A.; Nikolaeva, M. A.; Ushakova, I. V.; Lipatova, N. A.; Bozhedomova, G.
E.; Sukhikh, G. T. J. Reprod. Immunol. 2015, 112, 95–101.
(222) Huynh, T. Hum. Reprod. Update 2002, 8 (2), 183–198.
(223) Fraser, L. R. Hum. Reprod. 1998, 13 Suppl 1, 9–19.
(224) Breitbart, H. Mol. Cell. Endocrinol. 2002, 187 (1–2), 139–144.
(225) Smith, J. F.; Syritsyna, O.; Fellous, M.; Serres, C.; Mannowetz, N.; Kirichok, Y.; Lishko, P.
V. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (17), 6823–6828.
REFERENCES 209
(226) Cai, X.; Wang, X.; Clapham, D. E. Mol. Biol. Evol. 2014, 31 (10), 2735–2740.
(227) Alasmari, W.; Costello, S.; Correia, J.; Oxenham, S. K.; Morris, J.; Fernandes, L.;
Ramalho-Santos, J.; Kirkman-Brown, J.; Michelangeli, F.; Publicover, S.; Barratt, C. L. R.
J. Biol. Chem. 2013, 288 (9), 6248–6258.
(228) Darszon, A.; Acevedo, J. J.; Galindo, B. E.; Hernández-González, E. O.; Nishigaki, T.;
Treviño, C. L.; Wood, C.; Beltrán, C. Reproduction 2006, 131 (6), 977–988.
(229) Darszon, A.; Nishigaki, T.; Beltran, C.; Treviño, C. L. Physiol. Rev. 2011, 91 (4), 1305–
1355.
(230) Lishko, P. V; Botchkina, I. L.; Kirichok, Y. Nature 2011, 471 (7338), 387–391.
(231) Munire, M.; Shimizu, Y.; Sakata, Y.; Minaguchi, R.; Aso, T. J. Med. Dent. Sci. 2004, 51 (1),
99–104.
(232) Suarez, S. S.; Dai, X. Biol. Reprod. 1992, 46 (4), 686–691.
(233) Garcia, M. a; Meizel, S. Biol. Reprod. 1999, 60 (1), 102–109.
(234) Sánchez-Cárdenas, C.; Servin-Vences, M. R.; José, O.; Treviño, C. L.; Hernández-Cruz, A.;
Darszon, A. Biol. Reprod. 2014, 91 (August), 1–21.
(235) Clapham, D. E. Cell 2007, 131 (6), 1047–1058.
(236) Suarez, S. S.; Ho, H. C. Reprod. Domest. Anim. 2003, 38 (2), 119–124.
(237) Sumigama, S.; Mansell, S.; Miller, M.; Lishko, P. V.; Cherr, G. N.; Meyers, S. A.; Tollner,
T. Biol. Reprod. 2015, 93 (6), 130.
(238) Suarez, S. S. Hum. Reprod. Update 2008, 14 (6), 647–657.
(239) Suarez, S. S.; Ho, H. Reprod Dom Anim 2003, 124, 119–124.
(240) Suarez, S. S.; Katz, D. F.; Owen, D. H.; Andrew, J. B.; Powell, R. L. Biol. Reprod. 1991, 44
(2), 375–381.
(241) England, T. N. English J. 2002, 346 (10), 725–730.
REFERENCES 210
(242) Ortega, N. M.; Bosch, P. 2009.
(243) Bowdin, S.; Allen, C.; Kirby, G.; Brueton, L.; Afnan, M.; Barratt, C.; Kirkman-Brown, J.;
Harrison, R.; Maher, E. R.; Reardon, W. Hum. Reprod. 2007, 22 (12), 3237–3240.
(244) Neri, Q. V.; Tanaka, N.; Wang, A.; Katagiri, Y.; Takeuchi, T.; Rosenwaks, Z.; Palermo, G.
D. Minerva Ginecol. 2004, 56 (3), 189–196.
(245) Cox, G. F.; Bürger, J.; Lip, V.; Mau, U. A.; Sperling, K.; Wu, B.-L.; Horsthemke, B. Am. J.
Hum. Genet. 2002, 71 (1), 162–164.
(246) Hansen, M.; Kurinczuk, J. J.; Bower, C.; Webb, S. N. Engl. J. Med. 2002, 346 (10), 725–
730.
(247) Johnson, M. D. Fertil. Steril. 1998, 70 (3), 397–411.
(248) Good, R. J. J. Adhes. Sci. Technol. 1992, 6 (12), 1269–1302.
(249) Chen, W.; Fadeev, A. Y.; Hsieh, M. C.; Öner, D.; Youngblood, J.; McCarthy, T. J.
Langmuir 1999, 15 (10), 3395–3399.
(250) Wang, X.; Liu, L.-H.; Ramstrom, O.; Yan, M. Exp. Biol. Med. 2009, 234 (10), 1128–1139.
(251) Siegbahn, K.; Edvarson, K. Nucl. Phys. 1956, 1 (3), 137–159.
(252) Nascente, P. a. P. J. Mol. Catal. A Chem. 2005, 228, 145–150.
(253) Verma, H. R. At. Nucl. Anal. Methods 2007, 1–90.
(254) Hollander, J. M.; Jolly, W. L. Acc. Chem. Res. 1970, 3 (193).
(255) Wang, X.; Liu, L.-H.; Ramstrom, O.; Yan, M. Exp. Biol. Med. 2009, 234 (10), 1128–1139.
(256) Fadley, C. S. J. Electron Spectros. Relat. Phenomena 2010, 178–179 (C), 2–32.
(257) Drude, P. Ann. der Phys. und Chemie 1888, 270 (7), 489–531.
(258) Vedam, K. Thin Solid Films 1998, 313–314, 1–9.
(259) Habraken, F. H. P. M.; Gijzeman, O. L. J.; Bootsma, G. A. Surf. Sci. 1980, 96 (1–3), 482–
507.
REFERENCES 211
(260) Fujiwara, H. Spectroscopic Ellipsometry: Principles and Applications; John Wiley & Sons,
Ltd.: Tokyo, Japan, 2007.
(261) Fenstermaker, C. A.; McCrackin, F. L. Surf. Sci. 1969, 16, 85–96.
(262) De Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17 (7), 1759–1772.
(263) Tompkins, H. G. A User’s Guide to Ellipsometry; Elsevier Inc., 1993.
(264) Schubert, M. Ann. der Phys. 2006, 15 (7–8), 480–497.
(265) Loescher, D. H.; Detry, R. J.; Clauser, M. J. J. Opt. Soc. Am. 1971, 61 (9), 1230.
(266) Gonçalves, D.; Irene, E. A. Quim. Nova 2002, 25 (5), 794–800.
(267) Chiarini-Garcia, H.; Parreira, G. G.; Almeida, F. R. C. L. In Light Microscophy: Methods
and Protocols; 2011; pp 3–18.
(268) Light Microscopy; Chiarini-Garcia, H., Melo, R. C. N., Eds.; Methods in Molecular
Biology; Humana Press: Totowa, NJ, 2011; Vol. 689.
(269) Spring, K. R.; Davidson, M. W. Nikon Microsc. 2008.
(270) Lichtman, J. W.; Conchello, J.-A. Nat. Methods 2005, 2 (12), 910–919.
(271) In Fundamentals of Light Microscopy and Electronic Imaging; John Wiley & Sons, Inc.:
Hoboken, NJ, USA, 2012; pp 1–19.
(272) Buckman, J. F.; Hernandez, H.; Kress, G. J.; Votyakova, T. V.; Pal, S.; Reynolds, I. J. J.
Neurosci. Methods 2001, 104 (2), 165–176.
(273) Pendergrass, W.; Wolf, N.; Poot, M. Cytometry 2004, 61A (2), 162–169.
(274) Chikte, S.; Panchal, N.; Warnes, G. Cytom. Part A 2014, 85 (2), 169–178.
(275) Franke, W. W.; Appelhans, B.; Schmid, E.; Freudenstein, C.; Osborn, M.; Weber, K.
Differentiation 1979, 15 (1–3), 7–25.
(276) Willig, K. I.; Kellner, R. R.; Medda, R.; Hein, B.; Jakobs, S.; Hell, S. W. Nat. Methods 2006,
3 (9), 721–723.
REFERENCES 212
(277) Grant, E. R.; Dubin, A. E.; Zhang, S.-P.; Zivin, R. a; Zhong, Z. J. Pharmacol. Exp. Ther.
2002, 300 (1), 9–17.
(278) Jares-Erijman, E. A.; Jovin, T. M. Nat. Biotechnol. 2003, 21 (11), 1387–1395.
(279) Seiffert, S.; Oppermann, W. J. Microsc. 2005, 220 (1), 20–30.
(280) Reichert, W. M.; Truskey, G. A. J. Cell Sci. 1990, 96, 219–230.
(281) Axelrod, D. 2008; pp 169–221.
(282) Axelrod, D. 1989; pp 245–270.
(283) Liedberg, B.; Nylander, C.; Lundström, I. Biosens. Bioelectron. 1995, 10 (8), i–ix.
(284) Wood, R. W. Philos. Mag. Ser. 6 1912, 23 (134), 310–317.
(285) Wood, R. W. Philos. Mag. Ser. 6 1902, 4 (21), 396–402.
(286) Tudos, A. J.; Schasfoort, R. B. M. Handbook of Surface Plasmon Resonance; RSC
Publishing, 2008.
(287) Homola, J. Surface Plasmon Resonance Based Sensors; Homola, J., Ed.; Springer Series
on Chemical Sensors and Biosensors; Springer Berlin Heidelberg: Berlin, Heidelberg,
2006; Vol. 4.
(288) Piliarik, M.; Vaisocherová, H.; Homola, J. In Biosensors and Biodetection; Rasooly, A.,
Herold, K. E., Eds.; Humana Press: Totowa, NJ, 2009; pp 65–88.
(289) Piliarik, M.; Homola, J. Opt. Express 2009, 17 (19), 16505.
(290) Homola, J.; Yee, S. S.; Gauglitz, G. Sensors Actuators B Chem. 1999, 54 (1–2), 3–15.
(291) Boardman, A. D. Electromagnetic surfaces modes; John Wiley & Sons, 1982.
(292) Homola, J. Anal. Bioanal. Chem. 2003, 377 (3), 528–539.
(293) Ordal, M. A.; Long, L. L.; Bell, R. J.; Bell, S. E.; Bell, R. R.; Alexander, R. W.; Ward, C. A.
Appl. Opt. 1983, 22 (7), 1099.
(294) Homola, J. Chem. Rev. 2008, 108 (2), 462–493.
REFERENCES 213
(295) Liedberg, B.; Lundström, I.; Stenberg, E. Sensors Actuators B Chem. 1993, 11 (1–3), 63–
72.
(296) Liedberg, B.; Nylander, C.; Lunström, I. Sensors and Actuators 1983, 4, 299–304.
(297) Homola, J. Anal. Bioanal. Chem. 2003, 377 (3), 528–539.
(298) Zoski, C. Handbook of Electrochemistry; Elsevier Science, 2007.
(299) Gueshi, T.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. Interfacial Electrochem. 1978,
89 (2), 247–260.
(300) Slevin, C. J.; Macpherson, J. V.; Unwin, P. R. J. Phys. Chem. B 1997, 101 (50), 10851–
10859.
(301) Coles, B. A.; Compton, R. G.; Brett, C. M. A.; Brett, A. M. C. F. O. J. Electroanal. Chem.
1995, 381 (1–2), 99–104.
(302) Grieshaber, D.; MacKenzie, R.; Vörös, J.; Reimhult, E. Sensors 2008, 8 (3), 1400–1458.
(303) Kounaves, S. P. In Handbook of Instrumental Techniques for Analytical Chemistry;
Settle, F. A., Ed.; Prentice Hall PTR, 1997; pp 709–725.
(304) Nadjo, L.; Savéant, J. M. J. Electroanal. Chem. Interfacial Electrochem. 1973, 48 (1),
113–145.
(305) Settle, F. A. Handbook of instrumental techniques for analytical chemistry; Settle, F. A.,
Ed.; Prentice Hall PTR, 1997.
(306) Davies, T. J.; Compton, R. G. J. Electroanal. Chem. 2005, 585 (1), 63–82.
(307) Wang, H.; Chen, S.; Li, L.; Jiang, S. Langmuir 2005, 21 (7), 2633–2636.
(308) Andersen, H. C. J. Chem. Phys. 1980, 72 (4), 2384–2393.
(309) Allen, M. P.; Tidesley, D. J. Computer simulation of liquids; Oxford University Press,
1989.
(310) Materials Studio, version 4.0, Accelrys Inc., San Diego, 2006.; Accelerys Inc.: San Diego,
REFERENCES 214
2006.
(311) Frisch, M. J. et al. Gaussian 09 (Revision B.01), Gaussian, Inc., Wallingford CT.;
Gaussian, Inc., Wallingford CT., 2009.
(312) Nash, K.; Lefievre, L.; Peralta-Arias, R.; Morris, J.; Morales-Garcia, A.; Connolly, T.;
Costello, S.; Kirkman-Brown, J. C.; Publicover, S. J. J. Vis. Exp. 2010, No. 40.
(313) Amman, R. P.; Waberski, D. Theriogenology 2014, 81 (1), 5–17.
(314) Zareie, H. M.; Boyer, C.; Bulmus, V.; Nateghi, E.; Davis, T. P. ACS Nano 2008, 2 (4), 757–
765.
(315) Balamurugan, S.; Ista, L. K.; Yan, J.; López, G. P.; Fick, J.; Himmelhaus, M.; Grunze, M. J.
Am. Chem. Soc. 2005, 127 (42), 14548–14549.
(316) Young, D. D.; Deiters, A. ChemBioChem 2008, 9 (8), 1225–1228.
(317) Demirel, G. B.; Dilsiz, N.; Ergün, M. A.; Çakmak, M.; Çaykara, T. J. Mater. Chem. 2011,
21 (28), 10415.
(318) Mannix, R. J.; Kumar, S.; Cassiola, F.; Montoya-Zavala, M.; Feinstein, E.; Prentiss, M.;
Ingber, D. E. Nat. Nanotechnol. 2008, 3 (1), 36–40.
(319) Mu, L.; Liu, Y.; Zhang, S.; Liu, B.; Kong, J. New J. Chem. 2005, 29 (6), 847.
(320) Castner, D. G.; Ratner, B. D. Biomedical surface science: Foundations to frontiers; 2002;
Vol. 500.
(321) Soliman, M.; Nasanit, R.; Abulateefeh, S. R.; Allen, S.; Davies, M. C.; Briggs, S. S.;
Seymour, L. W.; Preece, J. A.; Grabowska, A. M.; Watson, S. A.; Alexander, C. Mol.
Pharm. 2012, 9 (1), 1–13.
(322) Stevenson, M.; Ramos-Perez, V.; Singh, S.; Soliman, M.; Preece, J. A.; Briggs, S. S.; Read,
M. L.; Seymour, L. W. J. Control. Release 2008, 130 (1), 46–56.
(323) Immoos, C. E.; Lee, S. J.; Grinstaff, M. W. J. Am. Chem. Soc. 2004, 126 (35), 10814–
REFERENCES 215
10815.
(324) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. 2003, 100 (16), 9134–9137.
(325) Huber, D. L. Science (80-. ). 2003, 301 (5631), 352–354.
(326) Yamanaka, H.; Yoshizako, K.; Akiyama, Y.; Sota, H.; Hasegawa, Y.; Shinohara, Y.; Kikuchi,
A.; Okano, T. Anal. Chem. 2003, 75 (7), 1658–1663.
(327) Yoshizako, K.; Akiyama, Y.; Yamanaka, H.; Shinohara, Y.; Hasegawa, Y.; Carredano, E.;
Kikuchi, A.; Okano, T. Anal. Chem. 2002, 74 (16), 4160–4166.
(328) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Langmuir
2007, 23 (18), 9409–9415.
(329) Palazon, F.; L??onard, D.; Mogne, T. Le; Zuttion, F.; Chevalier, C.; Phaner-Goutorbe, M.;
Souteyrand, ??liane; Chevolot, Y.; Cloarec, J. P. Beilstein J. Nanotechnol. 2015, 6 (1),
2272–2277.
(330) Ng, C. C. A.; Magenau, A.; Ngalim, S. H.; Ciampi, S.; Chockalingham, M.; Harper, J. B.;
Gaus, K.; Gooding, J. J. Angew. Chemie Int. Ed. 2012, 51 (31), 7706–7710.
(331) Curreli, M.; Li, C.; Sun, Y.; Lei, B.; Gundersen, M. A.; Thompson, M. E.; Zhou, C. J. Am.
Chem. Soc. 2005, 127 (19), 6922–6923.
(332) Yang, W.; Baker, S. E.; Butler, J. E.; Lee, C.; Russell, J. N.; Shang, L.; Sun, B.; Hamers, R. J.
Chem. Mater. 2005, 17 (5), 938–940.
(333) Crist, B. V.; Crist, B. V. Elements 1999, 1.
(334) Wang, X.; Liu, L.-H.; Ramstrom, O.; Yan, M. Exp. Biol. Med. 2009, 234 (10), 1128–1139.
(335) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. Handbook of X-ray
Photoelectron Spectroscopy; 1992.
(336) Tong, Y.; Tyrode, E.; Osawa, M.; Yoshida, N.; Watanabe, T.; Nakajima, A.; Ye, S.
Langmuir 2011, 27 (9), 5420–5426.
REFERENCES 216
(337) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111 (18), 7164–7175.
(338) Häussling, L.; Michel, B.; Ringsdorf, H.; Rohrer, H. Angew. Chemie Int. Ed. English 1991,
30 (5), 569–572.
(339) Fernandez-Torrente, I.; Monturet, S.; Franke, K. J.; Fraxedas, J.; Lorente, N.; Pascual, J. I.
Phys. Rev. Lett. 2007, 99 (17), 176103.
(340) Neff, J. L.; Söngen, H.; Bechstein, R.; Maass, P.; Kühnle, A. J. Phys. Chem. C 2015, 119
(44), 24927–24931.
(341) Spinke, J.; Liley, M.; Schmitt, F. ‐J.; Guder, H. ‐J.; Angermaier, L.; Knoll, W. J. Chem. Phys.
1993, 99 (9), 7012–7019.
(342) Barinaga-Rementeria Ramírez, I.; Mebrahtu, S.; Jergil, B. J. Chromatogr. A 2002, 971 (1–
2), 117–127.
(343) Hiller, Y.; Gershoni, J. M.; Bayer, E. A.; Wilchek, M. Biochem. J. 1987, 248 (1), 167–171.
(344) Holmberg, A.; Blomstergren, A.; Nord, O.; Lukacs, M.; Lundeberg, J.; Uhlén, M.
Electrophoresis 2005, 26 (3), 501–510.
(345) Dundas, C. M.; Demonte, D.; Park, S. Appl. Microbiol. Biotechnol. 2013, 97 (21), 9343–
9353.
(346) Casalini, S.; Dumitru, A. C.; Leonardi, F.; Bortolotti, C. A.; Herruzo, E. T.; Campana, A.;
de Oliveira, R. F.; Cramer, T.; Garcia, R.; Biscarini, F. ACS Nano 2015, 9 (5), 5051–5062.
(347) Adamczyk, M.; Moore, J. A.; Yu, Z. Methods 2000, 20 (3), 319–328.
(348) Ju, H. J. Anal. Test. 2017, 1 (1), 7.
(349) Mitchell, J. Sensors 2010, 10 (8), 7323–7346.
(350) Byrne, B.; Stack, E.; Gilmartin, N.; O’Kennedy, R. Sensors (Switzerland) 2009, 9 (6),
4407–4445.
(351) Carralero, V.; González-Cortés, A.; Yáñez-Sedeño, P.; Pingarrón, J. M. Anal. Chim. Acta
REFERENCES 217
2007, 596 (1), 86–91.
(352) Guiochon-Mantel, A.; Loosfelt, H.; Lescop, P.; Sar, S.; Atger, M.; Perrot-Applanat, M.;
Milgrom, E. Cell 1989, 57 (7), 1147–1154.
(353) Blackmore, P. F.; Im, W. Bin; Bleasdale, J. E. Mol. Cell. Endocrinol. 1994, 104 (2), 237–
243.
(354) Calogero, A. E.; Burrello, N.; Barone, N.; Palermo, I.; Grasso, U.; D’Agata, R. Hum.
Reprod. 2000, 15 (suppl 1), 28–45.
(355) Aurell, C.; Meizel, W. S. Dev. Biol. 1993, 159 (2), 679–690.
(356) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6 (3), 682–691.
(357) Dubois, L.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437–463.
(358) Rahman, M. S.; Kwon, W. S.; Pang, M. G. Biomed Res. Int. 2014, 2014.
(359) De Jonge, C. Hum. Reprod. Update 2005, 11 (3), 205–214.
(360) Xia, J.; Ren, D. Reprod. Biol. Endocrinol. 2009, 7 (1), 119.
(361) Lesaicherre, M.-L.; Uttamchandani, M.; Chen, G. Y. .; Yao, S. Q. Bioorg. Med. Chem.
Lett. 2002, 12 (16), 2079–2083.
(362) Spinola, S. M.; Cannon, J. G. J. Immunol. Methods 1985, 81 (1), 161–165.
(363) Böhme, U.; Scheler, U. Chem. Phys. Lett. 2007, 435 (4–6), 342–345.
(364) Jachimska, B.; Pajor, A. Bioelectrochemistry 2012, 87, 138–146.
(365) Mitchell, J. S.; Wu, Y. Biosensors 2010, No. February, 151–168.
(366) Omidfar, K.; Khorsand, F.; Darziani Azizi, M. Biosens. Bioelectron. 2013, 43 (1), 336–347.
(367) Breitbart, H. Cell. Mol. Biol. 2003, 49 (July), 321–327.
(368) Darszon, A.; Hernández-Cruz, A. Pflugers Arch. 2014, 466 (4), 819–831.
(369) Durán, S.; Duch, M.; Patiño, T.; Torres, A.; Penon, O.; Gómez-Martínez, R.; Barrios, L.;
Esteve, J.; Nogués, C.; Pérez-García, L.; Plaza, J. A. Sensors Actuators B Chem. 2015, 209,
REFERENCES 218
212–224.
(370) Wendeln, C.; Rinnen, S.; Schulz, C.; Kaufmann, T.; Arlinghaus, H. F.; Ravoo, B. J. Chem. -
A Eur. J. 2012, 18 (19), 5880–5888.
(371) Wendeln, C.; Rinnen, S.; Schulz, C.; Kaufmann, T.; Arlinghaus, H. F.; Ravoo, B. J. Chem. -
A Eur. J. 2012, 18 (19), 5880–5888.
(372) Vong, T.; ter Maat, J.; van Beek, T. A.; van Lagen, B.; Giesbers, M.; van Hest, J. C. M.;
Zuilhof, H. Langmuir 2009, 25 (24), 13952–13958.
(373) Rozkiewicz, D. I.; Brugman, W.; Kerkhoven, R. M.; Ravoo, B. J.; Reinhoudt, D. N. J. Am.
Chem. Soc. 2007, 129 (37), 11593–11599.
(374) Zhang, G.-J.; Tanii, T.; Zako, T.; Funatsu, T.; Ohdomari, I. Sensors Actuators B Chem.
2004, 97 (2–3), 243–248.
(375) Del Campo, A.; Boos, D.; Spiess, H. W.; Jonas, U. Angew. Chemie - Int. Ed. 2005, 44 (30),
4707–4712.
(376) Slocik, J. M.; Beckel, E. R.; Jiang, H.; Enlow, J. O.; Zabinski, J. S.; Bunning, T. J.; Naik, R. R.
Adv. Mater. 2006, 18 (16), 2095–2100.
(377) Li, Y.; Niehaus, J. C.; Chen, Y.; Fuchs, H.; Studer, A.; Galla, H.-J.; Chi, L. Soft Matter 2011,
7 (3), 861–863.
(378) Durán, S.; Duch, M.; Patiño, T.; Torres, A.; Penon, O.; Gómez-Martínez, R.; Barrios, L.;
Esteve, J.; Nogués, C.; Pérez-García, L.; Plaza, J. A. Sensors Actuators, B Chem. 2015,
209, 212–224.
(379) Mazia, D.; Schatten, G.; Sale, W. J. Cell Biol. 1975, 66 (1), 198–200.
(380) Kim, Y. H.; Baek, N. S.; Han, Y. H.; Chung, M.-A.; Jung, S.-D. J. Neurosci. Methods 2011,
202 (1), 38–44.
(381) Ooi, E. H.; Smith, D. J.; Gadêlha, H.; Gaffney, E. A.; Kirkman-Brown, J. R. Soc. Open Sci.
REFERENCES 219
2014, 1.
(382) Pantano, C. G. In Experimental Techniques of Glass Science; American Ceramic Society,
1993; pp 129–160.
(383) Hooper, A. E.; Werho, D.; Hopson, T.; Palmer, O. Surf. Interface Anal. 2001, 31 (9), 809–
814.
(384) Bozso, F.; Yates, J. T.; Choyke, W. J.; Muehlhoff, L. J. Appl. Phys. 1985, 57 (8), 2771.
(385) Brückner, R.; Chun, H.-U.; Goretzki, H.; Sammet, M. J. Non. Cryst. Solids 1980, 42 (1–3),
49–60.
(386) Serra, J.; González, P.; Liste, S.; Serra, C.; Chiussi, S.; León, B.; Pérez-Amor, M.; Ylänen,
H. O.; Hupa, M. J. Non. Cryst. Solids 2003, 332 (1–3), 20–27.
(387) WARD, C.; FRENCH, D. Fuel 2006, 85 (16), 2268–2277.
(388) Gross, T.; Ramm, M.; Sonntag, H.; Unger, W.; Weijers, H. M.; Adem, E. H. Surf. Interface
Anal. 1992, 18 (1), 59–64.
(389) Cerofolini, G. F.; Galati, C.; Renna, L. Surf. Interface Anal. 2003, 35 (12), 968–973.
(390) Wang, J.; Yang, X.; Hu, W.; Li, B.; Yan, J.; Hu, J. Chem. Commun. 2007, No. 46, 4931.
(391) Shircliff, R. A.; Stradins, P.; Moutinho, H.; Fennell, J.; Ghirardi, M. L.; Cowley, S. W.;
Branz, H. M.; Martin, I. T. Langmuir 2013, 29 (12), 4057–4067.
(392) Wang, Y.; Lieberman, M.; Hang, Q.; Bernstein, G. Int. J. Mol. Sci. 2009, 10 (2), 533–558.
(393) Yan, H.; Li, S.; Jia, Y.; Ma, X. Y. RSC Adv. 2015, 5 (17), 12578–12582.
(394) Hayashi, K.; Matsuda, T.; Takeyama, T.; Hino, T. Agric. Biol. Chem. 1966, 30 (4), 378–
384.
(395) Battocchio, C.; Porcaro, F.; Mukherjee, S.; Magnano, E.; Nappini, S.; Fratoddi, I.;
Quintiliani, M.; Russo, M. V.; Polzonetti, G. J. Phys. Chem. C 2014, 118 (15), 8159–8168.
(396) Weidner, T.; Bretthauer, F.; Ballav, N.; Motschmann, H.; Orendi, H.; Bruhn, C.;
REFERENCES 220
Siemeling, U.; Zharnikov, M. Langmuir 2008, 24 (20), 11691–11700.
(397) Martín-García, B.; Polovitsyn, A.; Prato, M.; Moreels, I. J. Mater. Chem. C 2015, 3 (27),
7088–7095.
(398) Muyldermans, S. Annu. Rev. Biochem. 2013, 82 (1), 775–797.
(399) Chakravarty, R.; Goel, S.; Cai, W. Theranostics 2014, 4 (4), 386–398.